Interleukin-1 receptor antagonists, compositions, and methods of treatment

ABSTRACT

Peptides that are designed to inhibit the biological activity of the IL-1R type 1 receptor and inhibit IL-1R/IL-1RacP related cell signaling and biological activity are disclosed. Compositions comprising IL-1R antagonists of the present invention are useful in the treatment of IL-1 related diseases or conditions such as arthritis, rheumatoid arthritis, osteoarthritis, and inflammatory bowel disease as well as other chronic or acute inflammatory diseases. This invention also discloses an isolated compound having an IL-1R antagonist activity, said compound being selected from the group consisting of: a peptide comprising the amino acid sequence RYTPELX, wherein R, Y, T, P, E, L, refer to their corresponding amino acids, and X is selected from no amino acid and alanine (A); and a derivative of (a) wherein the derivative incorporates one, two or three amino acid modification selected from an amino acid addition, deletion or substitution in the RYTPEL portion of the peptide, and wherein said derivative maintains its antagonist IL-1R activity.

FIELD OF THE INVENTION

The present invention relates to IL-1 receptor antagonists and methodsof modulating IL-1 receptors activity with same. More specifically, thepresent invention is concerned with extracellular, non-competitive IL-1receptor (IL1R/IL1RAcP) peptides and peptidomimetic antagonists, theiridentification and their therapeutic uses. More particularly, thepresent invention relates to peptide and peptidomimetic antagonists foruse in the treatment of IL-1 associated diseases such as rheumatoidarthritis and inflammatory bowel disease. The present invention hasapplication in the field of biochemistry and medicinal chemistry.

BACKGROUND OF THE INVENTION

Cytokines are generic terms for designating biologically activehormone-like proteins (interleukins, interferons, tumor necrosis factor,growth factors) that mediate their effects through a superfamily ofreceptors. Cytokines and their receptors constitute a powerful controlnetwork by which cells signal and coordinate cell proliferation anddifferentiation, cell death and survival. Cytokines can be specificallylow molecular weight peptides having very potent biological activity.Their mechanism of action is generally autocrine and paracrine and theyact ultimately by regulating gene expression.

The Interleukin-1 (IL-1) family of polypeptide hormones represent animportant class of cytokines which are expressed by a variety of celltypes including monocytes (which is the predominant source of IL-1),fibroblasts, endothelial cells, smooth muscle cells, osteoclasts,astrocytes, epithelial cells, T-cells, B-cells and numerous cancercells. This family of cytokines consists of more than 7 distinct butstructurally related molecules including IL-1α, IL-1β and IL-18, whichelicit a biologic response, and IL-1ra, a naturally produced receptorantagonist (Kumar, et al. 2000).

IL-1α and IL-1β genes are both located on chromosome 2. Each genecontains seven exons and are homologous in a region of the sixth exon.Both IL-1α and IL-1β are initially produced as 31 kDa precursors but areprocessed by proteases to produce the 17.5 kDa mature proteins.Receptors for IL-1 recognize both α and β forms and both forms havesimilar biological properties. IL-1α is the predominant form in micewhereas IL-1β is the predominant cytokine in human. The biologicalproperties of IL-1 are numerous and include mediating many immunologicaland inflammatory responses to infection and injury.

Despite its normally beneficial effects on an organism response toinfection and injury, circumstances have come to light in which theactions of IL-1 are harmful. For example, inappropriate production orresponse to IL-1 have been shown in many acute and chronic inflammatorydiseases such as rheumatoid arthritis, inflammatory bowel disease (IBD),osteoarthritis, psoriasis, septic shock, encephalitis and respiratorydistress syndrome. IL-1 has been shown to play a role in several otherillnesses including Alzheimer's disease, periventricular leukomalacia,meningitis, stroke, and a number of autoimmune diseases.

Interleukin-1 (IL-1) plays a primary upstream role in the regulation ofinflammation by stimulating generation of inflammatory mediators likeIL-6, prostaglandin E₂ (PGE₂; via the induction the COX-2 and PGEsynthase (mPGES) expression) and itself, therefore enhancing the processof inflammation. Another biological activity of IL-1 is to induceproliferation and activation of numerous cell types like T-cells(Cullinan, et al. 1998; Dunne and O'Neill 2003). IL-1 may also increasethe level of collagenase in an arthritic joint and has been implicatedin the acute and chronic stages of immunopathology in rheumatoidarthritis. IL-1 may be responsible for altering endothelial cellfunction, directing the chemotaxis of lymphocytes and leucocytes intosynovial tissue and inducing the secretion of latent collagenase bychondrocytes and fibroblasts. IL-1 is considered, along with TNF, as theprototype of inflammatory cytokines. However, the effects of IL-1 arenot limited to inflammation and this cytokine also plays a role in boneformation and remodeling, insulin secretion and fever induction.

As a major pro-inflammatory cytokine, IL-1 is a potentially powerfultarget for therapeutic intervention in diseases like articular cartilageinjury such as in arthritis. Osteoarthritis and rheumatoid arthritis areonly second to heart disease for causing work disabilities in NorthAmerica and their prevalence increase dramatically with age (Halleguaand Weisman 2002).

Two distinct receptors of IL-1 have been cloned and characterized:IL-1RI (Sims, et al. 1989), which generates the biological effects ofIL-1; and IL-1RII. In addition, a receptor accessory protein (IL-1RAcP), which is the putative signal-transducing subunit of the receptorcomplex, has been identified. IL-1R type I is found mainly on T cells,keratinocytes, fibroblasts, chondrocytes, synovicytes and epithelialcells. In order to generate a biological effect, IL-1R has to bind toIL-1 and subsequently to IL-1 RAcP to trigger signal transduction. Theextracellular portion of IL-1R contains three Ig-like domains that bindIL-1 (Vigers, et al. 1997; Vigers et al. 2000). As opposed to the IL-1Rreceptor subunit and according to studies involving antibodies againstextracellular portions of IL-1R accessory protein, the latter does notinteract with the cytokine (Cullinan et al. 1998; Laye et al. 1998;Malinowsky et al. 1998; Casadio et al. 2001).

The first event in signal transduction, following IL-1 binding, is theformation of an IL-1R/IL-1RacP complex which leads to IRAK (IL-1receptor associated kinase) recruitment to the complex and to a cascadeof phosphorylation by kinases, causing the activation of transcriptionalfactors including NFκB and AP-1. The IL-1R/IL-1RacP complex can alsorecruit and activate kinases like PI3K and Akt and can also lead to theactivation of the PLC/PKC pathway of signalization (Daun and Fenton,2000).

Two major clinical applications of IL-1R antagonists are the treatmentof arthritis and inflammatory bowel disease (IBD). The treatmentsavailable for these pathologies are currently limited. They often resultin toxicity and secondary effects. The demand in the medical world forsafer and more targeted therapies is therefore considerable.

The current approaches in the field of IBD and rheumatoid arthritistherapies include the development of soluble receptors, monoclonalantibodies directed against IL-1R and TNFR, mimetic of cytokines,antisense techniques and kinase inhibitors (Vigers et al. 1997; Vigerset al. 2000; Hallegua and Weisman 2002; Bouma and Strober 2003). In theparticular case of IL-1, a natural soluble receptor IL-1R^(a) mimetic,Anakinra, (generic name Kineret™) was developed by Amgen for treatmentof severely active rheumatoid arthritis in replacement of methotretaxe(an inhibitor of dihydrofolate (folic acid) reductase enzyme). In thecase of another major pro-inflammatory cytokine receptor, TNFR, twoantagonists, etanercept (Enbrel™, Amgen) and infliximab (Remicade™,Schering-Ploug), have also been developed.

Antagonists of the prior art are either competitive (e.g. solublereceptors, antibodies, cytokine mimetics), most often costly to produceor difficult to apply in vivo (e.g., antisense). Because the ligandexceeds by far the concentration of the receptor, the concentration ofcompetitive inhibitor needed to inhibit the interaction of IL-1 with itsnative receptor is often substantial.

Therefore, there remains a need for novel therapies that candown-regulate the activity mediated by IL-1.

The present invention seeks to meet these needs and other needs.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention thus concerns non-competitive, efficient andselective extracellular IL-1 receptor antagonists, which overcome one ormore of the drawbacks of the IL-1 receptor antagonists of the prior art.

The present invention also relates to the use of the non-competitive andselective antagonists of the present invention in the treatment of IL-1associated diseases.

In one embodiment, the compounds of the present invention are peptidesand peptidomimetics that inhibit the biological activity of IL-1R andinhibit cytokine activity by preventing signaling through the receptor.Thus, the inhibition of IL-1 mediated events leads for example, toanti-inflammatory responses, which are beneficial for the prophylaxis ortreatment of a variety of chronic and acute inflammatory diseases suchas rheumatoid arthritis and inflammatory bowel disease, among otherinflammatory diseases and diseases and conditions associated with IL-1function.

Designed to seek extracellular targets, unlike certain known drugcandidates which target intracellular regions of the IL-1 receptors, theantagonists of the present invention do not necessitate a priorpermeabilization or other disruption of cell membranes to gain access tothe target cell in order to produce a pharmacological response.

Because they function as non-competitive antagonists, a smaller amountof the antagonists of the present invention is necessary to inhibit thereceptor that they target, as compared to competitive inhibitors.

In a related embodiment, the antagonists of the present invention areadvantageously simple to synthesize.

The peptides, derivatives and peptidomimetics thereof of the presentinvention interact with a specific extracellular domain of theIL-1R/IL-1RacP receptor complex so as to inhibit the activity of thereceptor. Importantly, the peptides, peptide derivatives andpeptidomimetics of the present invention do not interact with the IL-1binding site on the IL-1R subunit and thus are considerednon-competitive peptide antagonists. The antagonists of the presentinvention are derived from the following API-101 sequence: APRYTVELA(SEQ ID NO:1) all with D-amino acids except where indicated (theasterisk in SEQ ID NO:135 indicates that the residue (R) is an L-aminoacid, see Table 1).

TABLE 1 LIST OF SEQUENCE NUMBERS SEQ ID NO: 1 API-101 APRYTVELA SEQ IDNO: 2 API-101.1 AARYTVELA SEQ ID NO: 3 API-101.2 APAYTVELA SEQ ID NO: 4API-101.3 APRATVELA SEQ ID NO: 5 API-101.4 APRYAVELA SEQ ID NO: 6API-101.5 APRYTA ELA SEQ ID NO: 7 API-101.6 APRYTVALA SEQ ID NO: 8API-101.7 APRYTVE AA SEQ ID NO: 9 API-101.9  PRYTVELA SEQ ID NO: 10API-101.10   RYTVELA SEQ ID NO: 11 API-101.11    YTVELA SEQ ID NO: 12API-101.12     TVELA SEQ ID NO: 13 API-101.101 XYTVELA (X = Citrulline)SEQ ID NO: 14 API-101.102 XYTVQ LA (X = Citrulline) SEQ ID NO: 15API-101.103 RYTVQLA SEQ ID NO: 16 API-101.104 RF TVELA SEQ ID NO: 17API-101.105 RYSVELA SEQ ID NO: 18 API-101.106 RYVVELA SEQ ID NO: 19API-101.107 RYTPELA SEQ ID NO: 20 API-101.108 RYTVEL SEQ ID NO: 21API-101.113 RYTPEL SEQ ID NO: 22 API-101.114 KYTPELA SEQ ID NO: 23API-101.115 XYTPELA (X = Ornithine) SEQ ID NO: 24 API-101.116 RW T P ELASEQ ID NO: 25 API-101.117 RYTPDLA SEQ ID NO: 26 API-101.118 RYT PQLA SEQID NO: 27 API-101.119 RYTPEFA SEQ ID NO: 28 API-101.120 RYTPEMA SEQ IDNO: 29 API-101.121 X RYT P ELA (X = Acetyl) SEQ ID NO: 30 API-101.122RYTPEPA SEQ ID NO: 31 API-101.123 RYTPA LA SEQ ID NO: 32 API-101.126XYTPEL (X = Ornithine) SEQ ID NO: 33 API-101.127 RFVP ELA SEQ ID NO: 34API-101.128 RW T PEL SEQ ID NO: 35 API-101.129 RYTPEV SEQ ID NO: 36API-101.132 RFTPEL SEQ ID NO: 37 API-101.133 KYTPEL SEQ ID NO: 38API-101.134 XYTPEL (X = Citrulline) SEQ ID NO: 39 API-101.135 *RYTPEL

Without being limited to a particular theory, IL-1 receptor antagonistsmay promote or stabilize a particular conformation of the IL-1 receptor,which results in inhibition, of the receptor activity. The peptides,peptide derivatives and peptidomimetics of the present invention inhibitIL-1 dependent intracellular signaling in a non-competitive way. Thesepeptides effectively prevent activation of the intracellular receptordomains responsible for IL-1 receptor signaling. Subsequent celltransduction events leading to expression of molecules (e.g.,inflammatory molecules like cytokines, cytokines receptors,prostaglandins, collagenase secretion . . . etc) responsible in part fordisease expression are thereby prevented.

These compounds include lead compounds and derivative compoundsconstructed so as to have the same or similar molecular structure orshape, as the lead compounds, but may differ from the lead compoundseither with respect to susceptibility to hydrolysis or proteolysis, orwith respect to their biological properties (e.g., increased affinityfor the receptor). The present invention also relates to compounds andcompositions that are useful for the treatment or prevention ofconditions, diseases or disorders associated with inappropriate IL-1production or IL-1 response.

In another embodiment, the present invention also relates topharmaceutical compositions comprising one or more of the compoundsdescribed herein and a physiologically acceptable carrier. Thesepharmaceutical compositions can be in a variety of forms including oraldosage forms, topic creams, suppository, nasal spray and inhaler, aswell as injectable and infusible solutions. Methods for preparingpharmaceutical composition are well known in the art as reference can bemade to Remington's Pharmaceutical Sciences, Mack Publishing Company,Eaton, Pa., USA.

The method of treatment of the present invention may be preventive andreduce the risk of developing an IL-1 associated disease or condition,and may be used to alleviate or obviate the condition. Theadministration of the therapeutic agent can be in any pharmaceuticallyacceptable form in a suitable carrier, and in therapeutically acceptabledose.

In view of the importance of IL-1 and or IL-1R/IL1RacP receptor functionin numerous pathways and conditions in animals, the present inventionhas a broad impact on the identification, validation and treatment ofconditions or diseases associated with IL-1 response (e.g., IL-1overexpression or abnormal signaling through IL-1R/IL1-RacP).

The present invention also concerns non-competitive, efficient andselective extracellular receptor agonists. In addition, the inventionrelates to the use of the non-competitive and selective agonists of thepresent invention in the treatment of interleukin-associated diseases inwhich the interleukin-1 (IL) already displays an inflammatory activityor other activity, one or more of an agonist of the present inventionincreasing the activity of the IL having the above-mentionedanti-inflammatory activity, or other activity. In a related embodiment,the agonists of the present invention are advantageously simple tosynthesize. Non-limiting examples of agonists in accordance with thepresent invention include peptides TTI-101.101 (SEQ ID NO:13) andTTI-101.102 (SEQ ID NO:14) as well as peptidomimetic TTI-101-137 andTTI-101-142 (FIGS. 29 and 30, respectively).

Non limiting examples of cytokines receptors for which agonists of thepresent invention can find a therapeutic use include:

-   -   (1) Pigment epithelium-derived factor. PEDF is synthetized by        retinal pigment epithelial cells and is an anti-angiogenic        factor in the retina. It also protects neurons from oxidative        stress and glutamate exotoxicity. An agonist of the present        invention would thus have a therapeutic potential in case of        abnormal neovascularization in the retina and in tumor growth        (e.g. diabetic retinopathy, retinopathy of prematurity, and        cancer; Barnstable et al. 2004).    -   (2) IL-4 receptor: IL4 is an anti-inflammatory cytokine that may        inhibit the production of inflammatory molecules like IL-1,        IL-6, TNF alpha by monocytes and TNF-alpha by T-cells at the        inflammatory site. IL-4 also inhibits the growth of colon and        mammary carcinomas. It also acts as an anti-inflammatory        cytokine in rheumatoid arthritis by a protective action on        chondrocytes and the inhibition of the production of        inflammatory mediators (Schuerwegh, et al).    -   (3) The IL-10 cytokines family: All of this family as a        beneficial effect on the inflammation site and its        anti-inflammatory effect has been described in the case of wound        healing, inflammatory bowel disease and psoriasis. IL-10        decreases the production of pro-inflammatory factors like IL-2,        TNF-alpha and IFN-gamma in Th1 cells. It decreases tumor growth        by inhibiting the infiltration of macrophages on tumor site (Li        et al. 2004; Asadullah et al. 2004).

In one embodiment the present invention relates to an isolated compoundselected from the group consisting of: a) a peptide, or isolated peptidewhich binds to IL-1R, or has an IL-1R antagonist activity (e.g.IL-1R/IL-1RacP antagonist activity), wherein the peptide or isolatedpeptide comprises the amino acid sequence RYTPELA, wherein R, Y, T, P,E, L, and A refer to their corresponding amino acids, and wherein saidpeptide can bind to IL-1R or has an IL-1R antagonist activity (e.g.IL-1R/IL-1RacP antagonist activity); and b) a derivative of (a) whereinthe derivative incorporates from one to four amino acid addition,deletion or substitution, and wherein the derivative competes with saidpeptide of (a) for binding to IL-1R or maintains its IL-1R antagonistactivity (e.g. IL-1R/IL-1RacP antagonist activity). In one particularembodiment, such derivative incorporates three, two or one amino acidaddition, deletion or substitution.

In one further embodiment, the present invention relates to a peptide,or isolated peptide which antagonizes the biological activity of IL-1Rwherein the peptide or isolated peptide comprises the sequencecharacterized by the general formula: RYTPELX, wherein R, Y, T, P, E,and L, refer to their corresponding amino acids, and wherein X isselected from no amino acid and alanine (A). The invention also relatesto derivatives of this general formula, wherein the derivativeincorporates one, two or three amino acid modification selected from anamino acid addition, deletion or substitution in the RYTPEL portion ofthe peptide RYTPELX, and wherein the derivative maintains its antagonistIL-1R activity. (e.g. IL-1R/IL-1RacP antagonist activity). Generally thesubstitution of an amino acid is made with a similar or conserved aminoacid, see below.

In one further embodiment, the derivative comprises two or less aminoacid modification selected from an amino acid addition, deletion orsubstitution in the RYTPEL portion of the peptide. In one particularembodiment such the peptide, isolated peptide or derivative thereof isselected from the group consisting of: 101-113, 101-103, 101-114,101-117, 101.10, 101.106, 101.116, 101.108, 101.135, 101.128, 101.9,101.105, 101.129, 101.11, 101.12, and 101.132. In yet another particularembodiment, the antagonist compounds are selected from the groupconsisting of: 101-113, 101-103, 101-114, 101-117, and 101.10.

In yet another embodiment, the present invention relates to an isolatedcompound having an IL-1R/IL1RacP antagonist activity, said compoundbeing selected from the group consisting of: a) a peptide comprising theamino acid sequence RYTPELX, wherein R, Y, T, P, E, L, refer to theircorresponding amino acids, and X is selected from no amino acid andalanine (A); and a derivative of (a) wherein the derivative incorporatesone, two or three amino acid modification selected from an amino acidaddition, deletion or substitution in the RYTPEL portion of the peptide,and wherein the derivative maintains its antagonist IL-1R/IL1RacPactivity. The invention of course also relates to such derivativeshaving only one, or only two such modification. Examples of suchantagonists comprise peptides 101-113, 101-103, 101-114, 101-117,101.10, 101.106, 101.116, 101.108, 101.135, 101.128, 101.9, 101.105,101.129, 101.11, 101.12, and 101.132, and more peptides 101-113,101-103, 101-114, 101-117, and 101.10.

In one specific embodiment, the present invention relates to a peptidewhich antagonizes the biological activity of IL-1R, wherein the peptidecomprises the sequence characterized by the general formula:

X-aa₁-aa₂-aa₃-aa₄-aa₅  Formula I

wherein X, aa₁, aa₂, aa₃-aa₄ and aa₅ are independently selected andwherein:

X is selected from A₁P₂R₃Y₄, A₁A₂R₃Y₄, A₁P₂A₃Y₄, A₁P₂R₃A₄, P₂R₃Y₄, R₃Y₄,Z₃Y₄, R₃F₄ and Y₄, wherein A, P, R, Y and F refer to their correspondingamino acids, the numbers refer to the positions of the amino acid in theA₁P₂R₃Y₄ sequence, and wherein Z is citrulline;

A₁ is selected from the group consisting of: alanine, leucine, valine,methionine, and φ, wherein φ defines an alpha-amino acid possessing ahydrophobic side-chain such as but not limited to: nor-leucine,iso-leucine, tert-leucine, cyclohexylalanine, allylglycine.

P₂ is selected from the group consisting of: proline, alanine,aminoisobutyric acid (Aib), N-Methyl-L-alanine (MeAla),trans-4-Hydroxyproline, diethylthiazolidine carboxylic acid (Dtc), andΩ, wherein Ω defines a conformational constraint-producing amino acid(Hanessian, S et al. 1997; Halab et al., 2000; Cluzeau and Lubell, 2004;Feng and Lubell 2001); non-limiting examples thereof include:azetidine-2-carboxylic acid, pipecolic acid, isonipecotic acid,4-(aminomethyl)benzoic acid, 2-aminobenzoic acid, nipecotic acid.

R₃ is selected from the group consisting of: histidine, lysine, alanine,ornithine, citrulline, 2-pyridylalanine, 3-pyridylalanine,4-pyridylalanine and arginine surrogates such as but not limited to4-amidinophenylacetyl, 4-amidinophenylpropionyl, 4-amidinophenylglycyl,4-amidinophenylmethylglycyl, 4-guanidinophenylacetyl,4-uanidinophenylpropionyl, 4-guanidinophenylglycyl,4-guanidinophenylmethylglycyl. (Masic and Kikelj, 2001; Feng and Lubell,2001)

Y₄ is selected from the group consisting of: no residue, phenylalanine,tryptophan, alanine, and Σ, wherein Σ defines an alpha-amino acidpossessing a hydrophobic side-chain Σ or aromatic side chain, examplesinclude but are not limited to: nor-leucine, iso-leucine, tert-leucine,cyclohexylalanine, allylglycine, naphthylalanine, pyridylalanine,histidine, tyrosine.

aa₁ is selected from the group consisting of: threonine, serine, valineand η, wherein η defines a neutral hydrophilic amino acid, examplesinclude but are not limited to, hydroxyvaline, beta,beta-dialkylserines,as described in Dettwiler and Lubell, 2004, homo-serine, allothreonine,hydroxyproline.

aa₂ is selected from the group consisting of: isoleucine, leucine,valine, proline, methionine, pipecolic acid, azetidine-2-carboxylicacid, hydroxyproline thiazolidine-a-carboxylic acid and φ, wherein φdefines an alpha-amino acid possessing a hydrophobic side-chain (seeabove).

aa₃ is selected from the group consisting of: aspartic acid, asparagine,glutamic acid, glutamine, serine, histidine, homoserine, beta-leucine,beta-phenylalanine, alpha amino adipic acid and T, wherein T defines a3-amino-5-phenylpentanoic acid-alpha-amino acid possessing a hydrophobicside-chain, an aromatic amine, an aliphatic amine and a primaryarylalkyl amine. Examples include but are not limited to benzylamine,phenylethylamine, 2,2-diphenylethylamine, 4-phenyl-benzylamine.

aa₄ is selected from: alanine, valine, isoleucine, leucine, methionine,phenylalanine, tryptophan and Λ, wherein Λ defines a neutral aliphaticamino acid. Examples include, but are not limited to, nor-leucine,iso-leucine, tert-leucine, cyclohexyalanine, allyglycine; an aliphaticamine of one to 10 carbons such as but not limited to methyl amine,iso-butylamine, iso-valerylamine, cyclohexylamine; an aromatic orarylalkylamine such as but not limited to aniline, naphtylamine,benzylamine, cinnamylamine, and phenylethylamine.

In another specific embodiment, the present invention relates to apeptide antagonist or derivative thereof, according to the presentinvention, wherein the antagonist is purified.

In yet another specific embodiment, the present invention relates to apeptide or derivative thereof, which antagonizes the biological activityof IL-1R, wherein the peptide or derivative thereof comprises thesequence characterized by one of the general formulas:

G₁-X-aa₁-aa₂-aa₃-aa₄-aa₅-  Formula II

-X-aa₁-aa₂-aa₃-aa₄-aa₅-G₂  Formula III

G₁-X-aa₁-aa₂-aa₃-aa₄-aa₅-G₂  Formula IV

wherein:

G₁ is attached to the amino-terminus of the peptide and is selected fromthe group consisting of: no residue, hydrogen, a straight chained orbranched alkyl group of one to eight carbons, an acyl group (RCO) (suchas acetyl, methyl, ethyl . . . ), propianoyl, butanoyl, iso-propianoyl,iso-butanoyl, or a tertiary amine (a dialkaylamino or monoalkylaminogroup).

G₂ is attached to the carboxy-terminus of the peptide and is selectedfrom the group consisting of: no residue hydrogen, NH₂, an aliphaticamine of one to ten carbons (such as but not limited to methyl amine),iso-butylamine, iso-valerylamine, cyclohexylamine, an aromatic amine orarylalkyl amine (such as but not limited to aniline, naphthylamine,benzylamine, cinnamylamine, phenylethylamine), and or a tertiary amine(a dialkaylamino or monoalkylamino group).

In yet another embodiment of the present invention, the peptide orderivative thereof, which antagonizes the biological activity of IL-1R,has the sequence characterized by one of the general formulas defined byFormula I, Formula II, Formula III or Formula IV.

In yet a further embodiment of the present invention the IL-1R/RacPpeptides or derivatives thereof, are relatively small molecules. In oneembodiment, the peptides have a size between 5 and 25 amino acids, moreparticularly between 5 and 16 amino acids, more particularly between 5and 10 amino acids, and even more particularly between 5 and 9 aminoacids.

In another specific embodiment, the present invention relates to apeptidomimetic derived from formulas I and IV which antagonize thebiological activity of IL-1R. In a more specific embodiment, thepeptidomimetics of the present invention are defined by the structuresrepresented in FIGS. 20 and 21.

In accordance with the present invention, there is provided apeptidomimetic antagonist of general sequence

R₁-aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-R₂

wherein R₁-aa₁, aa₂, aa₃, aa₄, aa₅, aa₆, aa₇, and R₂ are independentlyselected.

R₁ is selected from the group consisting of no residue, hydrogen, astraight chained or branched alkyl group of one to eight carbons, anacyl group (RCO—) wherein R is a straight chained or branched alkylgroup of one to eight carbons. Non-limiting examples of R include,methyl, ethyl, propyl, butyl, pentyl, iso-propyl, and iso-butyl.

aa₁ is selected from the group consisting of no residue, arginine,lysine, ornithine, citrulline, an omega-amino acyl group of two to eightcarbons, an omega guanidinyl acyl group of two to six carbons, anarginine surrogate, such as but not limited to 4-amidinophenylacetyl,4-amidinophenylpropionyl, 4-amidinophenylglycyl,4-amidinophenylmethylglycyl, 4-guanidinophenylacetyl,4-uanidinophenylpropionyl, 4-guanidinophenylglycyl,4-guanidinophenylmethylglycyl.

aa₂ is selected from the group consisting of no residue, tyrosine,phenylalanine, naphthylalanine, histidine, 4-hydroxyphenylglycine,tryptophan, phenylglycine, pyridylalanine, homoserine,3,4-dihydroxyphenylalanine, and 4-chlorophenylalanine.

aa₃ is selected from the group consisting of no residue, threonine,serine, beta-hydroxyvaline, allo-threonine, valine, tert-butylleucine,leucine, proline, pipecolic acid, azetidine-2-carboxylic acid,hydroxyproline, and alanine.

aa4 is selected from the group consisting of no residue, valine,proline, pipecolic acid, azetidine-2-carboxylic acid, hydroxyproline,thiazolidine-4-carboxylic acid, and2,2-dimethylthiazolidine-4-carboxylic acid.

In another embodiment aa₃-aa₄ together may consist of 3-aminoindolizidin-2-one 9-carboxylic acid, 3-amino pyrrolizidin-2-one8-carboxylic acid, 3-amino quinolizidin-2-one 10-carboxylic acid,8-amino indolizidin-9-one 2-carboxylic acid, a dipeptide surrogate orbeta-turn mimic such as but not limited to examples reviewed inHanessian et al.

aa₅ is selected from the group consisting of no residue, alanine,glutamic acid, glutamine, aspartic acid, asparagine, histidine,homoserine, beta-leucine, beta-phenylalanine, and alpha-amino adipicacid.

aa₆ is selected from the group consisting of no residue, alanine,valine, leucine, phenylalanine, tryptophan, an aliphatic amine of one toten carbons, such as but not limited to methyl amine, iso-butylamine,iso-valerylamine, cyclohexylamine, an aromatic or arylalkyl amine suchas but not limited to aniline, naphthylamine, benzylamine,cinnamylamine, or phenylethylamine.

aa₇ is selected from the group consisting of no residue, alanine,valine, leucine, phenylalanine, tryptophan, an aliphatic amine of one toten carbons, such as but not limited to methyl amine, iso-butylamine,iso-valerylamine, cyclohexylamine, an aromatic amine or arylalkyl aminesuch as but not limited to aniline, naphthylamine, benzylamine,cinnamylamine, or phenylethylamine.

R₂ is selected from the group consisting of no residue hydrogen, NH₂, analiphatic amine of one to ten carbons such as but not limited to methylamine, iso-butylamine, iso-valerylamine, cyclohexylamine, an aromaticamine and an arylalkyl amine such as but not limited to aniline,naphthylamine, benzylamine, cinnamylamine, phenylethylamine.

It should be noted that the stereochemical configurations of the chiralcenters of the residues in the general sequenceR₁-aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-R₂ can be of R- and S-, D- andL-configurations. In a preferred embodiment, the peptides consist of allD-isomers. Olefins can be of cis- and trans-geometry. Amino acidresidues in the general sequence R₁-aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-R₂ canalso be their aza-amino acid counter part in which the chiralalpha-carbon is replaced by nitrogen such as but not limited toaza-alanine, aza-tyrosine, aza-phenylalanine.

Although the present invention is examplified by the specificpeptidomimetics of FIGS. 20 and 21, as well as those exemplified inFIGS. 26 to 30, the present invention is not so limited. Based on thedisclosure herein, one skilled in the art can readily derivepeptidomimetics having antagonistic activity toward the IL-1 receptor,identify further IL-1R/IL-1RacP receptor inhibiting compounds or improvethose examplified herein. The peptidomimetics of the present inventionare less susceptible to degradation by endogenous proteases andtherefore have a longer half-life in vivo.

In one particular embodiment of the present invention, thepeptidomimetic having antagonistic activity toward the IL-1 receptor areselected from the group consisting of TTI-101.140, TTI-101.141,TTI-101.125, TTI-101.110, TTI-101.111, TTI-101.136 and TTI-101.143. In apreferred embodiment the peptidomimetics are selected from the groupconsisting of TTI-101.140, TTI-101.141, TTI-101.125, and TTI-101.110.

The compounds of the present invention are useful in vitro as uniquetools for understanding the biological role of IL-1 as well as the manyfactors thought to influence and be influenced by the production of IL-1and its binding to the IL-1R/IL-1RacP receptor. The antagonists of thepresent invention are also useful in the development of other compoundsthat bind the IL-1 receptor because the peptide antagonists of thepresent invention provide important information on the relationshipbetween structure and activity that will facilitate such development.

The antagonists of the present invention can also be used in assays asprobes for determining the expression of IL-1R receptor on the surfaceof cells. Such assays may be useful, for example, for determining thedegree of cellular inflammatory response to tissue infection or injury.Typically, the cells under study are exposed to the peptides orpeptidomimetics of the present invention, so as to enable them to bindto the receptors present on the cell surface, and reacted cells arevisualized (e.g., after wash, cell sorting, affinity chromatography,immunohistochemistry, autoradiography etc).

The compounds can be used as competitive inhibitors in assays to screenfor, or to characterize similar new peptide receptors antagonists. Insuch assays, as well as assays for determining IL-1R expression, thepeptides or peptidomimetics of the present invention can be used withoutmodification or they can be labeled (i.e., covalently or non-covalentlylinked to a moiety which directly or indirectly provide a detectablesignal). Examples of labels include radiolabels such as ¹²⁵I, ¹⁴C, and³H, enzymes such as alkaline phosphatase and horse radish peroxidase(U.S. Pat. No. 3,645,090), ligands such as biotin, avidin, luminescentcompounds including bioluminescent, phosphorescent, chemiluminescent orfluorescent labels (U.S. Pat. No. 3,940,475).

The compounds of the present invention can be administered to a subjectto completely or partially inhibit the effects of IL-1α or IL-1β on theIL-1R response in vivo. Thus the methods of the present invention areuseful in the therapeutic treatment of IL-1 related disorders. Forexample, the compositions of the present invention can be administeredin a therapeutically effective amount to treat symptoms related toinappropriate production of IL-1 or inappropriate response to IL-1(e.g., rheumatoid arthritis and inflammatory bowel disease).

In order to provide a clear and consistent understanding of terms usedin the specification and claims, including the scope to be given suchterms, a number of definitions are provided herein below.

DEFINITIONS

Unless defined otherwise, the scientific and technological terms andnomenclature used herein have the same meaning as commonly understood bya person of ordinary skill to which this invention pertains. Commonlyunderstood definitions of molecular biology terms can be found forexample in Dictionary of Microbiology and Molecular Biology, 2nd ed.(Singleton et al., 1994, John Wiley & Sons, New York, N.Y.), The HarperCollins Dictionary of Biology (Hale & Marham, 1991, Harper Perennial,New York, N.Y.), Rieger et al., Glossary of genetics: Classical andmolecular, 5^(th) edition, Springer-Verlag, New-York, 1991; Alberts etal., Molecular Biology of the Cell, 4^(th) edition, Garland science,New-York, 2002; and, Lewin, Genes VII, Oxford University Press,New-York, 2000. Generally, the procedures of cell cultures, infection,molecular biology methods and the like are common methods used in theart. Such standard techniques can be found in reference manuals such asfor example Sambrook et al. (2000, Molecular Cloning—A LaboratoryManual, Third Edition, Cold Spring Harbor Laboratories); and Ausubel etal. (1994, Current Protocols in Molecular Biology, John Wiley & Sons,New-York).

As used herein, the twenty natural amino acids and their abbreviationsfollow conventional usage. Stereoisomers (e.g., D-amino acids) such as□,□-disubstituted amino acids, N-alkyl amino acids, lactic acid andother unconventional amino acids may also be suitable components for thepolypeptides of the present invention. Examples of unconventional aminoacids include but are not limited to citrulline, ornithine, norvaline,4-(E)-butenyl-4(R)-methyl-N-methylthreonine (MeBmt), N-methyl-leucine(MeLeu), aminoisobutyric acid, statine, N-methyl-alanine (MeAla).

The term aromatic amines as used herein is understood as being amolecule having a ring of 6 to 10 carbon atoms and examples include butare not limited to phenylmethylamine, phenylethylamine,phenylpropylamine and an amine comprising saturated or unsaturatedhydrocarbon chain.

The term arylalkylamine as used herein is understood as being an aminecomprising a saturated or unsaturated hydrocarbon chain. A primaryarylalkylamine is composed of a ring of 6 to 10 carbon atoms andexamples include but are not limited to phenyl, tolyl, alkoxyphenyl,alkoxycarbonylphenyl and halophenyl.

The term “aryl” as used herein, is understood as being phenyl,1-naphthyl, and 2-naphthyl. The term “substituted aryl” as used herein,is understood as being phenyl, 1-naphthyl and 2-naphthyl having asubstituent selected from the group consisting of phenyl, heteroaryl,lower alkyl, lower alkoxy, lower alkylthio, halo, hydroxy,trifluoromethyl, amino, —NH(lower alkyl), and —N(lower alkyl)₂, as wellas being mono-, di- and tri-substituted phenyl, 1-naphthyl, and2-naphthyl comprising substituents selected from the group consisting ofmethyl, methoxy, methylthio, halo, hydroxy, and amino.

The term “alkyl” as used herein, is understood as being straight orbranched chain radicals having up to eight carbon atoms. The term “loweralkyl” as used herein, is understood as being straight or branchedradicals having up to four carbon atoms and is a preferred sub-groupingfor the term “alkyl”.

The term “substituted alkyl” as used herein, is understood as being suchstraight or branched chain radicals having up to 8 carbon atoms whereinone or more, preferably one, two, or three hydrogen atoms have beenreplaced by a substituent selected from the group consisting of hydroxy,amino, cyano, halogen, trifluoromethyl, —NH(lower alkyl), —N(loweralkyl)₂, lower alkoxy, lower alkylthio, and carboxy, aryl andheteroaryl.

Unless otherwise noted “IL-1” refers to either or both IL-1α and IL-1β.The term “IL” refers to the broad family of interleukins.

As mentioned above, as used herein, the twenty naturally occurringL-amino acids and their abbreviations follows conventional usage. In thepolypeptide notation used herein, the left-hand direction is theamino-terminal direction and the right-hand direction is thecarboxy-terminal direction, in accordance with standard usage andconvention. As used herein, the terms “peptides” and “polypeptides”refer to macromolecules which comprise a multiplicity of amino or iminoacids (or their equivalents) in peptide linkage, wherein thepolypeptides may comprise or lack posttranslational modifications.Therefore, the term peptides includes IL-1 receptor D-amino acidantagonists peptides and other modified forms of the peptides, so longas the modification does not alter its ability to modulate IL-1 receptoractivity. All antagonist peptides of the present invention share theability to modulate the activity of the IL-1 receptor. Non-limitingexamples of modifications include N-terminal acetylation, glycosylation,and biotinylation. Particularly modified versions of the peptidesaccording to the present invention are further described below.

The term “reverse-D peptide” refers herein to peptides containingD-amino acids, arranged in a reverse sequence relative to a peptidecontaining L-amino acids. Thus, the C-terminal residue of an L-aminoacid peptide becomes N-terminal for the D-amino acid peptide, and soforth. Reverse D-peptides may often retain the same tertiaryconformation and therefore the same activity, as the L-amino acidpeptides, but are more stable to enzymatic degradation in vitro and invivo, and thus have greater therapeutic efficacy than the originalpeptide (Brady and Dodson 1994; Jameson et al. 1994).

As used herein, the designation “functional derivative” denotes, in thecontext of a functional derivative of an amino acid sequence, a moleculethat retains a biological activity (either function or structural) thatis substantially similar to that of the original sequence. Thisfunctional derivative or equivalent may be a natural derivative or maybe prepared synthetically. Such derivatives include amino acid sequenceshaving substitutions, deletions, or additions of one or more aminoacids, provided that the biological activity of the protein is conserved(e.g. it acts as a non-competitive antagonist of IL-1 receptor). Thesubstituting amino acid generally has chemico-physical properties, whichare similar to that of the substituted amino acid. The similarchemico-physical properties include, similarities in charge, bulkiness,hydrophobicity, hydrophylicity and the like. The term “functionalderivatives” is intended to include “segments”, “variants”, “analogs” or“chemical derivatives” of the subject matter of the present invention.

The terms “biological activity” or “IL-1R/IL-1RacP activity” or“receptor activity” refers to any detectable biological activity of IL-1or IL-1R/IL-1RacP gene or protein. It can include specific biologicalactivity of IL-1R/IL-1RacP proteins in cell signaling. This includesmeasurement of PGE₂ production, proliferation assays and changes in geneand protein expression (e.g., IL-6, IL-1, COX enzymes). However,IL-1R/IL-1RacP activities are not limited to these important biologicalactivities. Biological activity also include for example, simple bindingto the IL-1R receptor with compounds, substrates, interacting proteinsand the like. For example, measuring the effect of a test compound onits ability to inhibit or increase (e.g., modulate) IL-1 response orIL-1R binding or interaction, is considered herein as measuring abiological activity of IL-1R according to the present invention. Broadlyintra- or inter-molecular binding of the receptor subunits (e.g., IL-1Rand IL-1RacP) in the absence vs the presence of the peptide, peptidederivative or peptidomimetic of the invention is yet another example ofa biological activity according to the invention. IL-1R/IL-1RacPbiological activity also includes any biochemical measurement of thisreceptor, conformational changes, phosphorylation status, any downstreameffect of the receptor's signaling such as protein phosphorylation (orany other posttranslational modification e.g. ubiquitination,sumolylation, palmytoylation, prenylation etc), kinase effect or anyother feature of the protein that can be measured with techniques knownin the art. Finally, IL-1R/IL-1RacP biological activity include adetectable change in cell architecture, cell proliferation or other cellphenotype that is modulated by the action of a ligand (i.e., 1L-1) onthe predetermined receptor.

The term “variant” refers herein to a protein, which is substantiallysimilar in structure and biological activity to the protein, to maintainat least one of its biological activities. Thus, provided that twomolecules possess a common activity and can substitute for each other,they are considered variants as that term is used herein, even if thecomposition, or secondary, tertiary or quaternary structure of onemolecule is not identical to that found in the other, or if the aminoacid sequence or nucleotide sequence is not identical. While the presentinvention relates to peptide sequences and their derivatives andvariants, it should be understood that nucleic acid sequences could bedesigned to express peptide antagonists of the present invention thatconsist of genetically encoded amino acids. Expression vectors,regulatory sequences (e.g. promoters), leader sequences and method togenerate same and introduce them in cells are well known in the art.Thus, in one embodiment, such antagonist peptides of the presentinvention are expressed in cells by recombinant technology. In oneembodiment the cells are prokaryotic cells and serve to produce andpurify such peptides. In another embodiment the eukaryotic cells arespecific eukaryotic cells in which IL-1 activity needs to be modulated.

The functional derivatives of the present invention can be synthesizedchemically or produced through recombinant DNA technology. All of thesemethods are well known in the art.

The term “subject” or “patient” as used herein refers to an animal,preferably a mammal, most preferably a human who is the object oftreatment, observation or experiment.

The terms “inhibiting,” “reducing” or “prevention,” or any variation ofthese terms, when used in the claims and/or the specification includesany measurable decrease or complete inhibition of the receptor activityto achieve a desired result. For example, a peptide is said to beinhibiting IL-1 activity when a decrease in PGE₂ production is measuredfollowing a treatment with the peptides, peptide derivatives orpeptidomimetics of the present invention as compared to in the absenceof these peptides.

As used herein, the term “purified” refers to a molecule (e.g. IL-1receptor, peptides, peptide derivatives, peptidomimetics, nucleic acids,proteins etc.) having been separated from a component of the compositionin which it was originally present. Thus, for example, a “purified IL-1receptor” has been purified to a level not found in nature. A“substantially pure” molecule is a molecule that is lacking in mostother components (e.g., 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, 100% free of contaminants). By opposition, the term “crude”means molecules that have not been separated from the components of theoriginal composition in which it was present. Therefore, the terms“separating” or “purifying” refers to methods by which one or morecomponents of the biological sample are removed from one or more othercomponents of the sample. Sample components include nucleic acids in agenerally aqueous solution that may include other components, such asproteins, carbohydrates, or lipids. A separating or purifying steppreferably removes at least about 70% (e.g., 70, 75, 80, 85, 90, 95, 96,97, 98, 99, 100%), more preferably at least about 90% (e.g., 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100%) and, even more preferably, at leastabout 95% (e.g., 95, 96, 97, 98, 99, 100%) of the other componentspresent in the sample from the desired component. For the sake ofbrevity, the units (e.g. 66, 67 . . . 81, 82, . . . 91, 92% . . . ) havenot systematically been recited but are considered, nevertheless, withinthe scope of the present invention.

The term “pharmaceutically acceptable carrier” refers to a carriermedium which does not interfere with the effectiveness of the biologicalactivity of the active ingredients of the compound and which is nottoxic for the host (e.g., patient) to whom it is administered.

“Therapeutically or pharmaceutically effective amount” refers herein tothe amount of composition of the present invention sufficient to inducea desired effect. Such result can be alleviation or reduction of thesigns, symptoms or causes of the disease or any other desired alterationof the target physiological system. For example, in the case ofinflammatory diseases (e.g., arthritis and inflammatory bowel disease) atypical result will involve decrease in inflammatory and immunologicalresponses.

As used herein, the terms “molecule”, “compound”, “agent” or “ligand”are used interchangeably and broadly to refer to natural, synthetic orsemi-synthetic molecules or compounds. The term “molecule” thereforedenotes for example chemicals, macromolecules, cell or tissue extracts(from plants or animals) and the like. Non-limiting examples ofmolecules include peptides, antibodies, carbohydrates and pharmaceuticalagents. The agents can be selected and screened by a variety of meansincluding random screening, rational selection and by rational designusing for example protein or ligand modeling methods such as computermodeling. The terms “rationally selected” or “rationally designed” aremeant to define compounds which have been chosen based on theconfiguration of interacting domains of the present invention or on theconfiguration of antagonist peptides and/or peptidomimetics of thepresent invention. As will be understood by the person of ordinaryskill, macromolecules having non-naturally occurring modifications arealso within the scope of the term “molecule”. For example,peptidomimetics, well known in the pharmaceutical industry and generallyreferred to as peptide analogs can be generated by modeling as mentionedabove. Similarly, in a preferred embodiment, the polypeptides of thepresent invention are modified to enhance their stability. It should beunderstood that in most cases this modification should not alter thebiological activity of the interaction domain. The molecules identifiedin accordance with the teachings of the present invention have atherapeutic value in diseases or conditions in which the physiology orhomeostasis of the cell and/or tissue is compromised by a defect in IL-1production or response. Non-limiting examples of such diseases orconditions include acute and chronic inflammatory diseases such asrheumatoid arthritis, inflammatory bowel disease (IBD), osteoarthritis,psoriasis, septic shock, encephalitis and respiratory distress syndrome.Alzheimer's disease, periventricular leukomalacia, meningitis, stroke,and a number of autoimmune diseases. It will be understood that thecompounds are herein described interchangeably as “API-X” “TTI-X” orsimply by the number of the compound (for example: “101.10”,“API-101.10” or “TTI-101.10”).

As used herein “antagonists”, “peptide antagonists” or “IL-1R/IL-1RacPantagonists” refers to any molecule capable of inhibiting (completely orpartially) a biological activity of IL-1 or IL-1R/IL-1RacP. The terms“antagonists”, “peptide antagonists” or “IL-1R/IL-1RacP antagonists”also include potentiators of known compounds with antagonist properties.

Herein the terminologies “mimic”, “mimetic”, peptidomimetic” and thelike are used herein interchangeably.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one” butit is also consistent with the meaning of “one or more”, “at least one”,and “one or more than one”.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or”.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, un-recitedelements or method steps.

The term “short peptide” is intended to mean a sequence of about 6-25amino acids.

As used herein, the term “purified” refers to a compound or compoundshaving been separated from a component of the composition in which itwas originally contained. Thus, for example, a “purified peptide” or a“purified composition of peptides” has been purified to a level notfound in nature. A “substantially pure” compound is a compound that islacking in most other components (e.g., 30, 40, 50, 60, 70, 75, 80, 85,90, 95, 96, 97, 98, 99, 100% free of contaminants). By opposition, theterm “crude” means compounds that have not been separated from thecomponents of the original composition in which it was present. For thesake of brevity, the units (e.g. 66, 67 . . . 81, 82, . . . 91, 92% . .. ) have not been specifically recited but are considered neverthelesswithin the scope of the present invention.

An “isolated peptide” or “isolated compound” is purified from itsnatural in vivo state, or state in which it is present with othercomponents at an earlier stage (from the synthesis for example).

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions and kits of theinvention can be used to achieve methods of the invention.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus, generally described the invention, reference will be madeto the accompanying drawings, showing by way of illustration only anillustrative embodiment thereof and in which:

FIG. 1 shows a proliferation assay on carcinoma cells (A549) in thepresence of IL-1β (10 ng/ml). Cells were preincubated with anti-IL-1RAPI-101 (SEQ ID NO: 1) peptide at different concentrations and thentreated with IL-1β at 10 ng/ml for 24 hours. ³H-thymidine was thenadded. After 24 hours cells were collected, lysed and ³H-thymidine wascounted.

FIG. 2 shows the inhibitory activity of anti-IL-1R peptides on IL-1induced PGE₂ synthesis by microvascular endothelial cells. Cells werepre-incubated 45 minutes with peptides and incubated with humanrecombinant IL-1β at a concentration of 10 ng/ml. PGE₂ synthesis wasdetermined in the growth medium.

FIG. 3 shows a dose response curve of PGE₂ synthesis with API-101 (SEQID NO: 1) peptide on microvascular endothelial cells in the presence of10 ng/ml of IL-1β.

FIG. 4 shows a dose response curve of vasodilatation of pialmicrovessels in the presence of 100 ng/ml of IL-1β and anti-IL-1βAPI-101 (SEQ ID NO: 1) and API-108 peptides.

FIG. 5 shows the in vitro PGE2 synthesis in the presence of IL-1β (10ng/ml) and alanine scanned peptides (10-6M) on microvascular pigendothelial cells (A) and on human chondrocytes (B).

FIG. 6 shows the ex vivo reversal of IL-1β-induced (100 ng/ml)vasodilatation of pial microvessels with the most active API-101 (SEQ IDNO: 1) alanine scanned peptides.

FIG. 7 shows a representation of the optimization scheme followed forAPI peptides.

FIG. 8 shows a proliferation assay performed on human lung fibroblasts(WI-38) in the presence of IL-1β (10 ng/ml) and truncated API-101 (SEQID NO: 1) derivatives.

FIG. 9 shows the cytotoxicity of derivatives of API-101 peptides (10⁻⁵M,MTT assay) on WI-38 cells (A); and brain microvascular endothelial cells(B).

FIG. 10 shows dose response curves of IL-1β (75 ng/ml) induced rat aortavasodilatation in presence of API-101 derivatives: API-101.10 (SEQ IDNO: 10) and API-101.12 (SEQ ID NO: 12) using a concentration range of10⁻⁵M to 10⁻¹⁰ M for both peptides.

FIG. 11 shows the in vivo effects of IL-1β—induced systemic hypotensionand serum PGE₂ synthesis by systemic administration of API-101 peptidesin rats. (A) Mean Blood Pressure (MBP) decrease in the presence of IL-1and/or peptide 101.10 (10⁻⁵M). (B) Modulation increase of serum PGE₂synthesis in presence of IL-1β and peptide 101.10 (10⁻⁵M).

FIG. 12 shows the effect of enteral injection of API-101.10 onIL-1β-induced hypotension and PGE₂ synthesis in rats. A) Mean BloodPressure (MBP) decrease in the presence of IL-1β and/or peptide 101.10.B) Modulation increase of serum PGE₂ synthesis in presence of IL-1β andpeptide 101.10.

FIG. 13 shows the sequences of API-101.10 peptide derivatives designedfor further optimization.

FIG. 14 shows the effect of API-101.10 peptide (systemic) in a rat modelof Inflammatory Bowel Disease (macroscopy). (A) Saline, (B) TNBS+Saline,and (C), TNBS+API-101.10 (2.2 mg/kg/day).

FIG. 15 shows the characterization of 101.10 peptide derivatives by Invitro peptide inhibition (IC₅₀ and maximum efficiency (Emax) are shown)of IL-1□-induced PGE2 synthesis in microvessels endothelial cells andWI-38 human fibroblasts.

FIG. 16 shows the therapeutic effect of API-101.10 in a rat model ofTNBS-induced inflammatory bowel disease (histology). (A) Saline, (B)TNBS (120 mg/ml)+Saline. (C) TNBS+API-101.10 (1.1 mg/kg/day).

FIG. 17 shows the characterization (inhibitory activity on IL-1>inducedPGE₂ production) of API 101 derivatives in porcine endothelial cells andchondrocytes.

FIG. 18 shows the characterization (IC₅₀ and maximum efficiency) ofAPI-101 derivatives.

FIG. 19 shows dose response assays for IL-1-induced PGE₂ synthesis inporcine microvascular endothelial cells in the presence of variouspeptidomimetics.

FIG. 20 shows the structure of the API-101.109 (“C2099”, ry(HyVal)pelaand API-1001-111 peptidomimetic.

FIG. 21 shows the structure of the API-101.110 peptidomimetic.

FIG. 22 shows the macroscopic evaluation of colonic injury in responseto intraperitoneal injections of peptides 101.10, 101.107 and 101.113 ina rat model of Inflammatory Bowel Disease.

FIG. 23 shows the effect of intraperitonal injected peptides 101.10,101.107 and 101.113 on tissue neutrophil infiltration (MPO assay) in arat model of Inflammatory Bowel Disease (48:00).

FIG. 24 shows the histological evaluation and scoring of tissue injuryin response to intraperitoneal injections of peptides 101.10, 101.107and 101.113 in a rat model of TNBS-induced Inflammatory Bowel Disease.

FIG. 25 shows the photographs of histological sections of colonictissues treated with TNBS and TTI-101.10 and 101.107. Panel A) Control(not treated); B) Animals treated with TNBS; C) Animals treated withTNBS and intraperitonal injections of 101.10 (1.0 mg/kg/d) peptide; D)Animals treated with intraperitonal injections of 101.107 (0.2 mg/kg/d).

FIG. 26 shows the structures and results of the characterization ofmimic derivatives of TTI-101.110

FIG. 27 shows the structures of mimic derivatives of TTI-101.125.

FIG. 28 shows the structures of other mimic derivatives of TTI-101.125.

FIG. 29 shows the structures and results of the characterization ofmimic derivatives of TTI-101.125.

FIG. 30 shows the structures and results of the characterization ofother mimic derivatives of TTI-101.125.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

One aim of the present invention is to describe a family of peptides andpeptidomimetic compounds capable of inhibiting the biological functionof IL-1 receptor accessory protein and therefore could be useful innumerous pathological conditions.

For purposes of clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into the followingsubsections:

I. Assays to Identify Peptides of the Present Invention II. PeptidePreparation III. Peptide Derivatives and Peptidomimetics IV. Assays toIdentify Peptidomimetics V. Pharmaceutical Compositions I. Assays toIdentify Peptides of the Present Invention

Methods for testing the ability of candidate compounds to inhibit IL-1receptor activity are presented herein. It will be understood that theinvention is not so limited. Indeed, other assays well known in the artcan be used in order to identify non-competitive, extracellular agonistsor antagonists of the present invention.

Generally, screens of a IL-1R/IL-1RacP antagonist (i.e., candidate ortest compounds or agents like peptides, peptidomimetics, small moleculeor other drugs) may be based on assays which measure a biologicalactivity of IL-1R/IL-1RacP. The assays of the present invention employeither a natural or recombinant IL-1 receptor. A cell fraction or cellfree screening assays for antagonists of IL-1 activity can use in situpurified, or purified recombinant IL-1 receptor. Cell-based assays canemploy cells which express IL-1 receptor naturally, or which containrecombinant IL-1 receptor. In all cases, the biological activity of IL-1receptor can be directly or indirectly measured; thus inhibitors oractivators of IL-1 receptor activity can be identified. The inhibitorsor activators themselves may be further modified by standardcombinatorial chemistry techniques to provide improved analogs of theoriginally identified compounds.

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a IL-1R/IL-1RacP receptor complex or biologically activeportion thereof, either natural or recombinant in origin, is contactedwith a test compound, and the ability of the test compound to modulateIL-1R/IL-1RacP receptor biological activity, e.g., modulation of PGE₂production, proliferation assays, binding of IL-1R to a binding partner(IL-1RacP) or any other measurable biological activity of the IL-1receptor is determined.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of IL-1R/IL-1RacP receptor complex canbe accomplished by determining the ability of the test compound tomodulate the activity of a downstream effector of a IL-1R/IL-1RacPreceptor target molecule. For example, the activity of the test compoundon the effector molecule can be determined. Non-limiting examples ofsuch downstream effector, include interleukin receptor activated kinase(IRAK); TRAF, activation of NI-KB (e.g. p65), mutagenic activatedprotein kinases (MAPK). Other examples of effector molecules which couldbe assayed to define the modulatory (agonist or antagonist) activity ofthe compounds of the present invention are described in Sims et al.2002; and Kashiwamura et al. 2002.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either IL-1, IL-1R,IL-1RacP or an interacting peptide or peptidomimetic of the presentinvention to facilitate separation of complexed from uncomplexed formsof one or both of the interacting proteins, as well as to accommodateautomation of the assay. Binding of a test compound to IL-1R protein orinteraction of IL-1R protein with a target molecule (e.g., IL-1RacP) inthe presence and absence of a candidate compound, can be accomplished inany vessel suitable for containing the reactants. Examples of suchvessels include microtiter plates, test tubes and micro-centrifugetubes. In one embodiment a fusion protein can be provided which adds adomain that allows one or both of the proteins to be bound to a matrix.For example. glutathione-S-transferase/IL-1R fusion proteins orglutathione-S-transferase/1L-1RacP fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.), orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or IL-1R protein and the mixture incubated underconditions conducive to complex formation (e.g. at physiologicalconditions for salt and pH). Following incubation the beads ormicrotiter plate wells are washed to remove any unbound components, andcomplex formation determined either directly or indirectly, for example,as described above. Alternatively, the complexes can be dissociated fromthe matrix, and the level of IL-1R binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices (which arewell-known in the art) can also be used in the screening assays of theinvention. For example, either a IL-1R protein or a IL-1R interactingmolecule (e.g., IL-1, IL-1RacP) can be immobilized utilizing conjugationof biotin and streptavidin. Biotinylated IL-1R protein or IL-1Rinteracting molecule can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with IL-1R protein or IL-1Rinteracting molecule, but which do not interfere with binding of theIL-1R protein to its IL-1R interacting molecule, can be derivatized tothe wells of the plate, and unbound target or IL-1R protein trapped inthe wells by antibody conjugation. Methods for detecting such complexes,in addition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theIL-1R protein or target molecule, as well as enzyme-linked assays whichrely on detecting an enzymatic activity associated with IL-1R receptoror IL-1R interacting molecule.

It shall be understood that the in vivo experimental models such asdescribed and exemplified herein can also be used to carry out an invitro assay.

In Vitro Assays

In one embodiment, candidate peptides are tested for their ability toactivate or inhibit IL-1 receptor's ability to modulate cellularproliferation with the incorporated tritiated thymidine method. In yetother embodiments, candidate peptides are tested for their ability toinhibit IL-1 receptor's ability to modulate cellular proliferation,using for example, the assays described in (Baker et al. 1995; Cheviron,Grillon et al. 1996); (Elliott et al. 1999; Hu et al. 1999).

In another embodiment, candidate peptides are tested for their abilityto modulate the phosphorylation state of IL-1R or portion thereof, or anupstream or downstream target protein in the IL-1R/IL-1RacP pathway,using for example an in vitro kinase assay.

In other embodiments, candidate peptides targeting IL-1R are tested forPGE₂ levels, IL-6 or collagenase expression or any other molecule havinga level which is modified following IL-1 stimulation in IL-1R/ILRacPexpressing cells, such as chondrocytes and fibroblasts.

In Vivo Assays

The assays described above may be used as initial or primary screens todetect promising lead compounds for further development. Lead peptideswill be further assessed in additional, different screens. Therefore,this invention also includes secondary IL-1R screens that may involvevarious assays utilizing mammalian cell lines expressing these receptorsor other assays.

Tertiary screens may involve the study of the identified inhibitors inanimal models for clinical symptoms. Accordingly, it is within the scopeof this invention to further use an agent (peptide or peptidomimetic)identified as described herein in an appropriate animal model such as arat or a mouse. For example, a peptide can be used in an animal model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatment (e.g.treatments of different types of disorders associated with aderegulation or malfunction of IL-1 receptor), as described herein.Non-limiting animal models which can be used in such assays include:collagen-induced arthritis in rat, animal model of acute IBD, tumorgrowth in immunosuppressed mouse, sensitization of the airways innewborn mice and any other known animal model including transgenicanimals.

II. Peptide Preparation

The peptide or peptide derivatives of the present invention are obtainedby any method of peptide synthesis known to those skilled in the art,including synthetic (e.g., exclusive solid phase synthesis, partialsolid phase synthesis, fragment condensation, classical solutionsynthesis) and recombinant techniques. For example, the peptides orpeptide derivatives can be obtained by solid phase peptide synthesis,which in brief, consists of coupling the carboxyl group of theC-terminal amino acid to a resin (e.g., benzhydrylamine resin,chloromethylated resin, hydroxymethyl resin) and successively addingN-alpha protected amino acids. The protecting groups maybe any suchgroups known in the art. Before each new amino acid is added to thegrowing chain, the protecting group of the previous amino acid added tothe chain is removed. Such solid phase synthesis has been described, forexample, by Merrifield, 1964, J. Am. Chem. Soc. 85: 2149; Vale et al.,1981, Science, 213: 1394-1397, in U.S. Pat. Nos. 4,305,872 and4,316,891, Bodonsky et al., 1966, Chem. Ind. (London), 38:1597; Piettaand Marshall, 1970, Chem. Comm. 650. The coupling of amino acids toappropriate resins is also well known in the art and has been describedin U.S. Pat. No. 4,244,946. (Reviewed in Houver-Weyl, Methods of OrganicChemistry. Vol E22a. Synthesis of Peptides and peptidomimetics, MurrayGoodman, Editor-in-Chief, Thieme. Stuttgart. New York 2002).

During any process of the preparation of the compound of the presentinvention, it may be necessary and/or desirable to protect sensitivereactive groups on any of the molecule concerned. This may be achievedby means of conventional protecting groups such as those described inProtective Groups In Organic Synthesis by T. W. Greene & P. G. M. Wuts,1991, John Wiley and Sons, New-York; and Peptides: chemistry and Biologyby Sewald and Jakubke, 2002, Wiley-VCH, Wheinheim p. 142. For example,alpha amino protecting groups include acyl type protecting groups (e.g.,trifluoroacetyl, formyl, acetyl), aliphatic urethane protecting groups(e.g., t-butyloxycarbonyl (BOC), cyclohexyloxycarbonyl), aromaticurethane type protecting groups (e.g., fluorenyl-9-methoxy-carbonyl(Fmoc), benzyloxycarbonyl (Cbz), Cbz derivatives) and alkyl typeprotecting groups (e.g., triphenyl methyl, benzyl). The amino acids sidechain protecting groups include benzyl (For Thr and Ser), Cbz (Tyr, Thr,Ser, Arg, Lys), methyl ethyl, cyclohexyl (Asp, His), Boc (Arg, His, Cys)etc. The protecting groups may be removed at a convenient subsequentstage using methods known in the art.

In one embodiment, the peptides of this invention, including the analogsand other modified variants, may generally be synthesized according tothe FMOC protocol in an organic phase with protective groups. They canbe purified with a yield of 70% with HPLC on a C18 column and elutedwith an acetonitrile gradient of 10-60%. Their molecular weight can thenbe verified by mass spectrometry (Reviewed in Fields, G. B. “Solid-PhasePeptide Synthesis”. Methods in Enzymology. Vol. 289, Academic Press,1997).

Alternatively, peptides of this invention that consist of geneticallyencoded amino acids may be prepared in recombinant systems usingpolynucleotide sequences encoding the peptides. It is understood that apeptide of this invention may contain more than one of theabove-described modifications within the same peptide. Also included inthis invention are pharmaceutically acceptable salt complexes of thepeptides of this invention or their derivatives.

Purification of the synthesized peptide or peptide derivatives iscarried out by standard methods, including chromatography (e.g., ionexchange, size exclusion, affinity), centrifugation, precipitation orany standard technique for the purification of peptides and peptidederivatives. In one embodiment, thin-layered chromatography is employed.In another embodiment, reverse phase HPLC is employed. Otherpurification techniques well known in the art and suitable for peptideisolation and purification may be used in the present invention.

Where the processes for the preparation of the compounds according tothe present invention give rise to mixtures of stereoisomers, theseisomers may be separated by conventional techniques such as preparativechromatography. The compounds may be prepared in racemic form, orindividual enantiomers may be prepared either by enantiospecificsynthesis or by resolution. The compounds may, for example, be resolvedinto their components enantiomers by standard techniques such as theformation of diastereoisomeric pairs by salt formation with an opticallyactive acid followed by fractional crystallization and regeneration ofthe free base. The compounds may also be resolved by formation ofdiastereomeric esters or amides, followed by removal of the chiralauxiliary. Alternatively, the compounds may be resolved using chiralHPLC column.

III. Peptide Derivatives and Peptidomimetics

In addition to peptides consisting only of naturally occurring aminoacids, peptidomimetics or peptide analogs are also encompassed by thepresent invention. Peptide analogs are commonly used in thepharmaceutical industry as non-peptide drugs with properties analogousto those of the template peptide. The types of non-peptide compounds aretermed “peptide mimetics” or peptidomimetics (Fauchere, J. 1986, Adv.Drug Res. 15: 29; Evans et al., 1987, J. Med. Chem. 30: 1229). Peptidemimetics that are structurally related to therapeutically usefulpeptides may be used to produce an equivalent or enhanced therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto paradigm polypeptide (i.e., a polypeptide that has a biological orpharmacological activity) such as naturally occurring receptor-bindingpolypeptides but have one or more peptide linkages optionally replacedby linkages like —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans),—CH₂SO—, —CH(OH)CH₂—, —COCH₂— etc., by methods well known in the art(Spatola A. F., Peptide Backbone Modifications, Vega Data, March 1983,1(3): 267; Spatola et al., Life Sci., 1986, 38:1243-1249; Hudson D. etal., Int. J. Pept. Res. 1979., 14: 177-185; Weinstein. B., 1983,Chemistry and Biochemistry, of Amino Acids, Peptides and Proteins,Weinstein Eds, Marcel Dekker, New-York,). Such peptide mimetics may havesignificant advantages over natural polypeptides including moreeconomical production, greater chemical stability, enhancedpharmacological properties (e.g., half-life, absorption, potency,efficiency etc), reduced antigenicity and others.

While peptides are effective in inhibiting wild-type IL-1 in vitro,their effectiveness in vivo might be compromised by the presence ofproteases. Serum proteases have specific substrate requirements. Thesubstrate must have both L-amino acids and peptide bonds for cleavage.Furthermore, exopeptidases, which represent the most prominent componentof the protease activity in serum, usually act on the first peptide bondof the peptide and require a free N-terminus (Powell et al. 1993), Inlight of this, it is often advantageous to utilize modified versions ofpeptides also termed peptide analogs or derivatives. The modifiedpeptides retain the structural characteristics of the original L-aminoacid peptides that confer biological activity with regard to IL-1, butare advantageously not readily susceptible to cleavage by proteaseand/or exopeptidases.

Systematic substitution of one or more amino acids of a consensussequence with D-amino acid of the same type (e.g., D-lysine in place ofL-lysine) may be used to generate more stable peptides. Thus, a peptidederivative or peptidomimetic of the present invention may be all L, allD or mixed D, L peptide. In a preferred embodiment, the peptides consistof all D-amino acids. The presence of an N-terminal or C-terminalD-amino acid increases the in vivo stability of a peptide sincepeptidases cannot utilize a D-amino acid as a substrate (Powell et al.1993). Reverse-D peptides are peptides containing D-amino acids,arranged in a reverse sequence relative to a peptide containing L-aminoacids. Thus, the C-terminal residue of an L-amino acid peptide becomesN-terminal for the D-amino acid peptide, and so forth. ReverseD-peptides retain the same tertiary conformation and therefore the sameactivity, as the L-amino acid peptides, but are more stable to enzymaticdegradation in vitro and in vivo, and thus have greater therapeuticefficacy than the original peptide (Brady and Dodson 1994; Jameson etal. 1994). In addition to reverse-D-peptide, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods well known in the art(Rizo et Gierasch, Ann. Rev. Biochem., 1992., 61: 387), for example, byadding cysteine residues capable of forming disulfide bridges whichcyclize the peptide. Cyclic peptides have no free N- or C-termini. Thus,they are not susceptible to proteolysis by exopeptidases, although theyare of course susceptible to endopeptidases, which do not cleave atpeptide termini. Thus, the amino acid sequences of the peptides withN-terminal or C-terminal D-amino acids and of the cyclic peptides areusually identical to the sequences of the peptides to which theycorrespond, except for the presence of N-terminal or C-terminal D-aminoacid residue, or their circular structure, respectively.

A cyclic derivative containing intramolecular disulfide bond may beprepared by conventional solid phase synthesis while incorporatingsuitable S-protected cysteine or homocysteine residues at the positionsselected for cyclization such as the amino and carboxy termini (SahM etal., 1996., J. Pharm. Pharmacol. 48: 197). Following completion of thechain assembly, cyclization can be performed either (1) by selectiveremoval of the S-protecting group with a consequent on-support oxidationof the corresponding two free SH-functions, to form a S—S bonds,followed by conventional removal of the product from the support andappropriate purification procedure; or (2) by removal of the peptidefrom the support along with complete side chain deprotection, followedby oxidation of the free SH-functions in highly dilute aqueous solution.

The cyclic derivative containing an intramolecular amide bond may beprepared by conventional solid phase synthesis while incorporatingsuitable amino and carboxyl side chain protected amino acid derivatives,at the position selected for cyclization. The cyclic derivativescontaining intramolecular —S— alkyl bonds can be prepared byconventional solid phases while incorporating an amino acid residue witha suitable amino-protected side chain, and a suitable S-protectedcysteine or homocysteine residue at the position selected forcyclization.

Substitution of unnatural amino acids for natural amino acids in asubsequence of the peptides can also confer resistance to proteolysis.Such a substitution can, for instance, confer resistance to proteolysisby exopeptidases acting on the N-terminus. Such substitutions have beendescribed and these substitutions do not affect biological activity.Examples of non-naturally occurring amino acids includeα,α-disubstituted amino acids, N-alkyl amino acids, lactic acids,C-α-methyl amino acids, and β-methyl amino acids. Amino acids analogsuseful in the present invention may include but are not limited toβ-alanine, norvaline, norleucine, 4-aminobutyric acid, orithine,hydroxyproline, sarcosine, citrulline, cysteic acid, cyclohexylalanine,2-aminoisobutyric acid, 6-aminohexanoic acid, t-butylglycine,phenylglycine, o-phosphoserine, N-acetyl serine, N-formylmethionine,3-methylhistidine and other unconventional amino acids. Furthermore, thesynthesis of peptides with unnatural amino acids is routine and known inthe art.

One other effective approach to confer resistance to peptidases actingon the N-terminal or C-terminal residues of a peptide is to add chemicalgroups at the peptide termini, such that the modified peptide is nolonger a substrate for the peptidase. One such chemical modification isglycosylation of the peptides at either or both termini. Certainchemical modifications, in particular N-terminal glycosylation, havebeen shown to increase the stability of peptides in human serum (Powellet al., 1993). Other chemical modifications which enhance serumstability include, but are not limited to, the addition of an N-terminalalkyl group, consisting of a lower alkyl of from 1 to 20 carbons, suchas an acetyl group, and/or the addition of a C-terminal amide orsubstituted amide group. In particular the present invention includesmodified peptides consisting of peptides bearing an N-terminal acetylgroup and/or a C-terminal amide group.

Also included by the present invention are other types of peptidederivatives containing additional chemical moieties not normally part ofthe peptide, provided that the derivative retains the desired functionalactivity of the peptide. Examples of such derivatives include (i) N-acylderivatives of the amino terminal or of another free amino group,wherein the acyl group may be an alkanoyl group (e.g., acetyl, hexanoyl,octanoyl), an aroyl group (e.g., benzoyl) or a blocking group such asF-moc (fluorenylmethyl-O—CO—); (ii) esters of the carboxy terminal or ofanother free carboxy or hydroxyl group; (iii) amide of thecarboxyterminal or of another free carboxyl group produced by reactionwith ammonia or with a suitable amine; (iv) phosphorylated derivatives;(v) derivatives conjugated to an antibody or other biological ligand andother types of derivatives.

Longer peptide sequences which result from the addition of extra aminoacid residues to the peptides of the invention are encompassed by thepresent invention since they should have the same biological activity(inhibit activation of IL-1 receptor) as the peptides described above.While peptides having a substantial number of additional amino acids arenot excluded, it will be recognized that some large polypeptides mayassume a configuration that masks the effective sequence, therebypreventing binding to IL-1R. These derivatives could act as competitiveantagonists and are thereby excluded from the invention. Thus, while thepresent invention encompasses peptides or derivatives having anextension, such longer peptides should be selected as not destroying themodulating activity of the peptide or derivative.

Other derivatives included in the present invention are dual peptidesconsisting of two of the same, or two different peptides of the presentinvention covalently linked to one another either directly or through aspacer, such as by a short stretch of alanine residues or by a putativesite for proteolysis (e.g., by cathepsin, see U.S. Pat. No. 5,126,249and European Patent No. 495,049). Multimers of the peptides of thepresent invention consist of polymer of molecules formed from the sameor different peptides or derivatives thereof.

In another embodiment, the peptide derivatives of the present inventionare chimeric or fusion proteins comprising a peptide of the presentinvention or fragment thereof linked at its amino or carboxy terminalend, or both, to an amino acid sequence of a different protein. Such achimeric or fusion protein may be produced by recombinant expression ofa nucleic acid encoding the protein. In one embodiment such a chimericor fusion protein contains at least 6 amino acids of a peptide of thepresent invention and has a functional activity equivalent or greater tothat of a peptide of the invention.

Peptide derivatives of the present invention can be made by altering theamino acid sequences by substitutions, additions or deletions thatprovide for functionally equivalent molecules, or functionally enhancedor diminished molecules, as desired. The derivative of the presentinvention include, but are not limited to those containing, as primaryamino acid sequence, all or part of the amino acid sequence of thepeptides of the present invention including altered sequences in whichfunctionally equivalent amino acid residues are substituted for anequivalent in the sequence. For example, one or more amino acid residueswithin the sequence can be substituted by another amino acid of asimilar polarity, which act as a functional equivalent, resulting in asilent alteration. Substitution for an amino acid within the sequencemay be selected from other members of the class to which the amino acidbelongs. For example, the positively charged (basic) amino acidsinclude, arginine, lysine and histidine. The nonpolar (hydrophobic)amino acids include, leucine, isoleucine, alanine, phenylalanine,valine, proline, tryptophan and methionine. The uncharged polar aminoacids include serine, threonine, cysteine, tyrosine, asparagine andglutamine. The negatively charged (acid) amino acids include glutamicacid and aspartic acid. The amino acid glycine is sometimes included inthe nonpolar amino acids family and sometimes in the uncharged (neutral)polar amino acids family. Substitutions that are done within a family ofamino acids are generally understood to be conservative substitutions.

In one particular embodiment of the present invention the antagonistpeptide comprises the sequence TTI-101.140, TTI-101.141, TTI-101.125,TTI-101.110, TTI-101.111, TTI-101.136 and TTI-101.143.

IV. Assays to Identify Peptidomimetics

As mentioned above, non-peptidyl compounds generated to replicate thebackbone geometry and pharmacophore display (peptidomimetics) of thepeptides identified by the methods of the present invention oftenpossess attributes of greater metabolic stability, higher potency,longer duration of action and better bioavailability.

The peptidomimetic compounds of the present invention can be obtainedusing any of the numerous approaches in combinatorial library methodsknown in the art, including: biological libraries; spatially addressableparallel solid phase or solution phase libraries; synthetic librarymethods requiring deconvolution; the ‘one-bead one-compound’ librarymethod; and synthetic library methods using affinity chromatographyselection. The biological library approach is limited to peptidelibraries, while the other four approaches are applicable to peptide,non-peptide oligomer or small molecule libraries of compounds (Lam,Anticancer Drug Des. 12: 145, 1997). Examples of methods for thesynthesis of molecular libraries can be found in the art, for examplein: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA. 90:6909; Erb et al.(1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994), J.Med. Chem. 37:2678; Cho et al. (1993) Science 261: 1303; Carell et al.(1994) Angew. Chem., Int. Ed Engl. 33:2059; and ibid 2061; and in Gallopet al. (1994) Med. Chem. 37:1233. Libraries of compounds may bepresented in solution (e.g. Houghten (1992) Biotechniques 13:412-421) oron beads (Lam (1991) Nature 354; 82-84), chips (Fodor (1993) Nature 364;555-556), bacteria or spores (Ladner U.S. Pat. No. 5,223,409), plasmids(Cull et al., (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or onphage (Scott and Smith (1990); Science 249:386-390). Examples of methodsfor the synthesis of molecular libraries can be found in the art, forexample in: DeWitt et al. (1993) supra; Erb et al. (1994) supra;Zuckerman et al. (1994) supra; Cho et al. (1993) supra; Carrell et al.(1994) Supra, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

Once a peptide of the present invention is identified, it may beisolated and purified by any number of standard methods including butnot limited to differential solubility (i.e., precipitation),centrifugation, chromatography (affinity, ion exchange, size exclusionand the like) or by any other standard techniques used for thepurification of peptides, peptidomimetics or proteins. The functionalproperties of an identified peptide of interest may be evaluated usingany functional assay known in the art. In one embodiment assays forevaluating downstream receptor function in intracellular signaling areused (e.g., PGE₂ synthesis).

In one embodiment, the peptidomimetic compounds of the present inventionare obtained with the following three-phase process: 1) scanning thepeptides of the present invention to identify regions of secondarystructure necessary for recognition and activity toward the IL-1receptor; 2) using conformationally constrained dipeptide surrogates torefine the backbone geometry and provide organic platforms correspondingto these surrogates; and 3) using the best organic platforms to displayorganic pharmocophores in libraries of candidates designed to mimic thedesired activity of the native peptide. In more details the three phasesare as follows. In phase 1, the peptide leads are scanned and theirstructure abridged to identify the requirements for their activity. Aseries of peptide analogs of the original are synthesized. In phase 2,the best peptide analogs are investigated using the conformationallyconstrained dipeptide surrogates. Indolizidin-2-one, indolizidin-9-oneand quinolizidinone amino acids (I²aa, I⁹aa and Qaa respectively) areused as platforms for studying backbone geometry of the best peptidecandidates. These and related plateforms (Reviewed in Halab, Li;Gosselin, F; Lubell, W D; Biopolymers (Peptide Science) Vol 55, 101-122.2000; Hanessian, S. J. McNaughton-Smith G; Lombart, H-G.; Lubell, W. D.Tetrahedron Vol. 53, 12789-12854, 1997) may be introduced at specificregions of the peptide in order to orient the pharmacophores indifferent directions. Biological evaluation of these analogs identifiesimproved leads that mimic the geometric requirements for activity. Inphase 3, the platforms from the most active leads are used to displayorganic surrogates of the pharmacophores responsible for activity of thenative peptide. The pharmacophores and scaffolds are combined in aparallel synthesis format. Of course derivation of peptides and thedifferent phases therefore, can be done by other means and methods knownin the art.

Structure function relationships determined from the peptides, peptidederivatives, peptidomimetics or other small molecules of the presentinvention may be used to refine and prepare analogous molecularstructures having similar or better properties. Thus, also within thescope of the present invention are molecules, in addition to thosespecifically disclosed, that share the structure, polarity, chargecharacteristics and side chain properties of the specific embodimentsexemplified herein.

In conclusion, the peptides, peptide derivatives, peptidomimetics orother small molecules of the present invention are functionally active(i.e., capable of exhibiting one or more of the identified functionalactivities associated with a peptide of the present invention). Forexample, such peptides, peptide derivatives, peptidomimetics or analogsthat inhibit a desired property (e.g., binding of the IL-1RacP to aprotein partner or ligand) can be used as inhibitors of such propertyand its physiological correlates. Peptides, derivatives, peptidomimeticsor analogs of the peptides of the present invention can be tested forthe inhibition of cell signaling through the IL-1R/IL-1RacP receptor byany functional assay known in the art (e.g., PGE₂ synthesis).

V. Pharmaceutical Compositions

The present invention relates to a method for inhibiting IL-1 receptoractivity through its interaction with the peptides, peptide derivativesand peptidomimetics of the present invention. In view of the importanceof IL-1 and or IL-1R/IL1RacP receptor function in numerous pathways andconditions in animals, the peptides, peptide derivatives andpeptidomimetics of the present invention are useful in the treatment ofconditions or diseases associated with IL-1 response (e.g., IL-1overexpression or abnormal signaling through IL-1R/IL1-RacP).

Therefore, methods of the present invention comprise administering to asubject in need thereof or at risk of being in need thereof an effectiveamount of a peptide, peptide derivative or peptidomimetic, or acomposition comprising a peptide, peptide derivative or peptidomimeticto a subject, to inhibit IL-1R/RacP biological activity. In oneembodiment, an effective amount of a therapeutic composition comprisinga peptide or peptide derivative thereof and a suitable pharmaceuticalcarrier is administered to a subject to inhibit IL-1R/IL-1RacPbiological activity to prevent, ameliorate symptoms or treat a disorder,disease or condition related to abnormal signaling throughIL-1R/IL-1RacP (e.g., overestimulation of the IL-1/IL-1RacP receptor viaan overproduction of IL-1/IL-1RacP ligand or via a constitutively activereceptor or any other defect). In one embodiment, the subject is ananimal. In another embodiment, the subject is a mammal, and preferably ahuman.

The peptides, peptide derivatives and peptidomimetics of the presentinvention are used in the treatment, prophylaxis or amelioration ofsymptoms in any disease condition or disorder where the inhibition ofIL-1R/IL-1RacP biological activity might be beneficial. Diseases,conditions or disorders to which the peptides, peptide derivatives orpeptidomimetics of the present invention may be beneficial include, butare not limited to the following examples: chronic and acuteinflammation diseases like rheumatoid arthritis, inflammatory boweldisease, septic shock, osteoarthritis, psoriasis, encephalitis,glomerulonephritis, respiratory distress syndrome and Reiter's syndrome.Other conditions include, systemic lupus erythematosus, scleroderma,Crohn's disease, ulcerative colitis, inflammatory joint disease,cachexia in certain leukemias, Alzheimer's disease, numerous types ofcancers, juvenile diabetes mellitus, pulmonary hypertension, stroke,periventricular leucopenia and meningitis.

Composition within the scope of the present invention should contain theactive agent (e.g. peptide, peptide derivative or peptidomimetic) in anamount effective to achieve the desired therapeutic effect whileavoiding adverse side effects. Pharmaceutically acceptable preparationsand salts of the active agent are within the scope of the presentinvention and are well known in the art. For the administration ofpolypeptide antagonists and the like, the amount administered should bechosen so as to avoid adverse side effects. The amount of thetherapeutic or pharmaceutical composition which is effective in thetreatment of a particular disease, disorder or condition will depend onthe nature and severity of the disease, the target site of action, thepatient's weight, special diets being followed by the patient,concurrent medications being used, the administration route and otherfactors that will be recognized by those skilled in the art. The dosagewill be adapted by the clinician in accordance with conventional factorssuch as the extent of the disease and different parameters from thepatient. Typically, 0.001 to 100 mg/kg/day will be administered to thesubject. Effective doses may be extrapolated from dose response curvesderived from in vitro or animal model test systems. For example, inorder to obtain an effective mg/kg dose for humans based on datagenerated from rat studies, the effective mg/kg dosage in rat is dividedby six.

Various delivery systems are known and can be used to administerpeptides, peptide derivatives or peptidomimetics or a pharmaceuticalcomposition of the present invention. The pharmaceutical composition ofthe present invention can be administered by any suitable routeincluding, intravenous or intramuscular injection, intraventricular orintrathecal injection (for central nervous system administration),orally, topically, subcutaneously, subconjunctivally, or via intranasal,intradermal, sublingual, vaginal, rectal or epidural routes.

Other delivery system well known in the art can be used for delivery ofthe pharmaceutical compositions of the present invention, for examplevia aqueous solutions, encapsulation in microparticles, ormicrocapsules.

In yet another embodiment, the pharmaceutical compositions of thepresent invention can be delivered in a controlled release system. Inone embodiment polymeric materials can be used (see Smolen and Ball,Controlled Drug Bioavailability, Drug product design and performance,1984, John Wiley & Sons; Ranade and Hollinger, Drug Delivery Systems,pharmacology and toxicology series, 2003, 2^(nd) edition, CRRC Press),in another embodiment, a pump may be used (Saudek et al., 1989, N. Engl.J. Med. 321: 574).

Compounds of the present invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The compounds of the present invention may alsobe coupled to a class of biodegradable polymers useful in achievingcontrolled release of the drug, non-limiting examples, include:polylactic acid, polyorthoesters, cross-linked amphipathic blockcopolymers and hydrogels, polyhydroxy butyric acid andpolydihydropyrans.

As mentioned above, pharmaceutical compositions of the present inventioncomprise a peptide, peptide derivative or peptidomimetic combined with apharmaceutically acceptable carrier. The term carrier refers todiluents, adjuvants, excipients such as a filler or a binder, adisintegrating agent, a lubricant a silica flow conditioner astabilizing agent or vehicles with which the peptide, peptide derivativeor peptidomimetic is administered. Such pharmaceutical carriers includesterile liquids such as water and oils including mineral oil, vegetableoil (e.g., peanut oil, soybean oil, sesame oil, canola oil), animal oilor oil of synthetic origin. Aqueous glycerol and dextrose solutions aswell as saline solutions may also be employed as liquid carriers of thepharmaceutical compositions of the present invention. Of course, thechoice of the carrier depends on the nature of the peptide, peptidederivative or peptidomimetic, its solubility and other physiologicalproperties as well as the target site of delivery and application. Forexample, carriers that can penetrate the blood brain barrier are usedfor treatment, prophylaxis or amelioration of symptoms of diseases orconditions (e.g. inflammation) in the central nervous system. Examplesof suitable pharmaceutical carriers are described in Remington: TheScience and Practice of Pharmacy by Alfonso R. Gennaro, 2003, 21^(th)edition, Mack Publishing Company.

Further pharmaceutically suitable materials that may be incorporated inpharmaceutical preparations of the present invention include absorptionenhancers, pH regulators and buffers, osmolarity adjusters,preservatives, stabilizers, antioxidants, surfactants, thickeners,emollient, dispersing agents, flavoring agents, coloring agents andwetting agents.

Examples of suitable pharmaceutical excipients include, water glucose,sucrose, lactose, glycol, ethanol, glycerol monostearate, gelatin, rice,starch flour, chalk, sodium stearate, malt, sodium chloride and thelike. The pharmaceutical compositions of the present invention can takethe form of solutions, capsules, tablets, creams, gels, powderssustained release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides (see Remington: The Science and Practice of Pharmacy byAlfonso R. Gennaro, 2003, 21^(th) edition, Mack Publishing Company).Such compositions contain a therapeutically effective amount of thetherapeutic composition, together with a suitable amount of carrier soas to provide the form for proper administration to the subject. Theformulations are designed so as to suit the mode of administration andthe target site of action (e.g., a particular organ or cell type).

The pharmaceutical compositions of the present invention can beformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those that form with free amino groups and those that react withfree carboxyl groups. Non-toxic alkali metal, alkaline earth metal andammonium salts commonly used in the pharmaceutical industry includesodium, potassium, lithium, calcium, magnesium, barium, ammonium, andprotamine zinc salts, which are prepared by methods well known in theart. The term also includes non-toxic acid addition salts, which aregenerally prepared by reacting the compounds of the present inventionwith suitable organic or inorganic acid. Representative salts includethe hydrobromide, hydrochloride, valerate, oxalate, oleate, laureate,borate, benzoate, sulfate, bisulfate, acetate, phosphate, tysolate,citrate, maleate, fumarate, tartrate, succinate, napsylate salts and thelike.

Examples of fillers or binders that may be used in accordance with thepresent invention include acacia, alginic acid, calcium phosphate(dibasic), carboxymethylcellulose, carboxymethylcellulose sodium,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, dextrin, dextrates, sucrose, tylose,pregelatinized starch, calcium sulfate, amylose, glycine, bentonite,maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodiumphosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose,guar gum, liquid glucose, compressible sugar, magnesium aluminumsilicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone,sodium alginate, tragacanth microcrystalline cellulose, starch, andzein. Another most preferred filler or binder consists ofmicrocrystalline cellulose.

Examples of disintegrating agents that may be used include alginic acid,carboxymethylcellulose, carboxymethylcellulose sodium,hydroxypropylcellulose (low substituted), microcrystalline cellulose,powdered cellulose, colloidal silicon dioxide, sodium croscarmellose,crospovidone, methylcellulose, polacrilin potassium, povidone, sodiumalginate, sodium starch glycolate, starch, disodium disulfite, disodiumedathamil, disodium edetate, disodiumethylenediaminetetraacetate (EDTA)crosslinked polyvinylpyrollidines, pregelatinized starch, carboxymethylstarch, sodium carboxymethyl starch, microcrystalline cellulose.

Examples of lubricants include calcium stearate, canola oil, glycerylpalmitostearate, hydrogenated vegetable oil (type I), magnesium oxide,magnesium stearate, mineral oil, poloxamer, polyethylene glycol, sodiumlauryl sulfate, sodium stearate fumarate, stearic acid, talc and, zincstearate, glyceryl behapate, magnesium lauryl sulfate, boric acid,sodium benzoate, sodium acetate, sodium benzoate/sodium acetate (incombination), DL leucine.

Examples of silica flow conditioners include colloidal silicon dioxide,magnesium aluminum silicate and guar gum. Another most preferred silicaflow conditioner consists of silicon dioxide.

Examples of stabilizing agents include acacia, albumin, polyvinylalcohol, alginic acid, bentonite, dicalcium phosphate,carboxymethylcellulose, hydroxypropylcellulose, colloidal silicondioxide, cyclodextrins, glyceryl monostearate, hydroxypropylmethylcellulose, magnesium trisilicate, magnesium aluminum silicate,propylene glycol, propylene glycol alginate, sodium alginate, carnaubawax, xanthan gum, starch, stearate(s), stearic acid, stearicmonoglyceride and stearyl alcohol.

The present invention also provides for modifications of peptides orpeptide derivatives such that they are more stable once administered toa subject (i.e., once administered it has a longer half-life or longerperiod of effectiveness as compared to the unmodified form). Suchmodifications are well known to those skilled in the art to which thisinvention pertain (e.g., polyethylene glycol derivatization a.k.a.PEGylation, microencapsulation, etc).

The IL-1R/IL-1RacP antagonists of the present invention may beadministered alone or in combination with other active agents useful forthe treatment, prophylaxis or amelioration of symptoms of a IL-1,IL-1R/IL-1RacP associated disease or condition. Thus, the compositionsand methods of the present invention can be used in combination withother agents exhibiting the ability to modulate IL-1 activity (e.g.,synthesis, release and/or binding to IL-1R/IL-1RacP) or to reduce thesymptoms of an IL-1 associated disease (e.g., rheumatoid arthritis andinflammatory bowl disease). Example of such agents include but are notlimited to antirheumatic drugs such as chloroquine, auranofin(Ridaura™), dexamethasone, sodium aurothiomalate, methotrexate (see Leeet al., 1988, Proc. Int. Acad. Sci, 85:1204), probucol (see Ku et al.,1988, Am. J. Cardiol. 62:778), pentoxyfylline (e.g., Sullivan et al.,1988, Infect. Immun. 56:1722), disulfuram (see Marx 1988, Science,239:257), antioxidants such as nordihydroguaiaretic acid (lee et al.,1988, Int J. Immunopharm., 10:385), IL-1 Trap (see e.g., 2003, Curr.Opin. Inv. Drugs, 4(5): 593-597), Anakinra (Kineret™, PCT ApplicationWO00236152), leflunomide, corticosteroids (Medrol™, Deltasone™,Orasone™) as well as other agents such as those described in Bender andLee (1989) Annual Reports in Medicinal Chemistry, chapter 20:Pharmacological Modulation of IL-1: 185-193). Other drugs may also beused in combination with the compounds of the present invention likeanti-inflammatory drugs such as Non Steroidal Antiinflammatory Drugs(NSAIDS, e.g., Rofecoxib (VIOXX™), Celecoxib (Celebrex™), Valdecoxib(Bextra™), Aspirin™, Advil™), anti TNF-α drugs (Infliximab, etanercept,adalimumab), collagenase inhibitors and others. Of course a combinationof two or more peptides, derivatives and peptide mimetics and theircombination with one or more drug can also be used, in all combinations(e.g. one or more peptide with one or more mimetic, one or more mimeticwith one or more derivative, one or more peptide with one or more drugetc.).

The present invention is illustrated in further details by the followingnon-limiting examples. The examples are provided for illustration onlyand should not be construed as limiting the scope of the invention.

EXAMPLE 1 Effect of API-101 Peptide

One example of the efficacy of the compounds and methods of the presentinvention is represented by the results obtained with the identifiedpeptide API-101 (SEQ ID NO 1: APRYTVELA).

All peptides described in the following examples have been synthesizedaccording to the FMOC protocol of solid phase synthesis in an organicphase with protective groups. They have been purified with a yield of70% with HPLC on a C18 column and eluted with an acetonitrile gradientof 10-60%. Their molecular weight have been verified by massspectrometry. Of course as alluded above, when natural amino acids areused, they can be obtained by genetic engineering techniques as known inthe art.

The proliferation effect of IL-1 was measured in A549 carcinoma cells inthe presence of peptide API-101 (SEQ ID NO: 1) and of IL-1 (10 ng/ml)using the incorporation of tritiated thymidine method. A549 cells werepre-incubated (45 min.) with different concentrations of peptides priorto stimulation with IL-1β (10 ng/ml) (37° C.) for 24 h; fetal calf serumwas omitted 24 h prior to stimulation with IL-1β to avoid proliferativeeffects by other mitotic agents. ³H-thymidine (1 μCi/ml) was then addedfor 24 h, after which cells were washed three times with 10% cold TCAand lysed with 0.1 N NaOH/0.1% Triton X-100. Radioactivity was measuredwith a scintillation counter. Experiments were repeated 3 times induplicates. As seen in FIG. 1, the peptide completely abrogated IL-1inhibition of proliferation with an IC₅₀ of 10⁻⁶M.

It is well known in the art that IL-1 induces the synthesis ofprostaglandin E₂ (PGE₂) in endothelial cells and/or chondrocytes invitro. Therefore, human chondrocytes and piglet brain microvascularendothelial cells were pre-incubated 45-60 minutes at 37° C. withpeptide API-101 (¹⁰⁻⁶M) and human IL-1 (10 ng/ml) was added in thegrowing medium. After 24 hours of incubation, growth medium wascollected and evaporated. PGE₂ measurement was determined by RIA assaywith a commercial kit (Cederlane).

FIG. 2 shows that PGE₂ synthesis induced by IL-1 (10 ng/ml) wasremarkably decreased by more than 50% with API-101 (SEQ ID NO: 1) at aconcentration of 10⁻⁶M with an IC₅₀ of 10⁻⁶ M (FIG. 3).

Ex Vivo Characterization

In another experiment, vasomotricity studies were performed on pigletpial vessels to further evaluate the particular effect of API-101 (SEQID NO: 1) on the vasodilator effect of IL-1β.

Ex vivo vasomotor response on brain vessels was performed as described(Li et al., 1996; Hou et al., 2000; Hou et al., 2001; Hou et al., 2003).Brains were removed from Yorkshire piglets. Slices of cortex exposingthe pial vessels were pinned to a wax base of a 20 ml bath containingKrebs buffer (pH 7.4) equilibrated with 95% O₂, 5% CO₂ and maintained at37° C. Microvessels were visualized and recorded using a video cameramounted on a dissecting microscope. Vascular diameter was measured usinga digital image analyzer. Vascular diameter was recorded before andafter topical application of pre-constricting agent, U46619 (10⁻⁷ M).After stabilization of preparation, the ligand (IL-1β, 75 ng/ml) wasadded until stable vasodilation was detected. Peptides were subsequentlyadded at different concentrations from 10⁻¹⁰ to 10⁻⁵ M. Reversal ofvasodilatation was visualized and measured as previously described (Hou,et al. 2000; Hou et al. 2001; Hou et al. 2003); triplicate measurementswere conducted on 2 animals.

As seen on FIG. 4, API-101 (SEQ ID NO: 1) could prevent the vasodilationinduced by IL-1β (75 ng/ml) with an IC₅₀ of 182 nM.

In summary, these results show that by targeting the IL-1R/IL-1Racpreceptor, API-101 (SEQ ID NO: 1) was efficient in inhibiting thebiological response of IL-1.

EXAMPLE 2 Effect of Derivatives of API-101 Obtained by Alanine Scan

Having demonstrated a significant effect of the API-101 (SEQ ID NO: 1)antagonist, experiments were carried out to provide structure functionrelationship data for API-101 and derivatives, to identify the mostimportant regions for activity. Alanine scan mutations were thereforeperformed on API-101 (SEQ ID NO: 1) (see FIG. 17 for the sequence of thepeptides). Of course, other amino acids could have been used in theplace of alanine to perform the scanning experiment.

In Vitro Characterization

A table summary of the results depending on the mutations performed isshown in FIG. 17.

Efficiencies and inhibitory activities of the mutated peptides weredetermined by measuring the inhibition of IL-1-induced PGE₂ synthesis(see experimental protocol above in Example 1). API-101.1 (SEQ ID NO: 2)only had a slightly improved efficacy in endothelial cells and inchondrocytes as compared to the parent peptide API-101 (SEQ ID NO: 1).On the other hand, API-101.5 (SEQ ID NO: 6), −101.6 (SEQ ID NO:7), and−101.7 (SEQ ID NO:8) lost almost all activity in both cell typessuggesting that the targeted VELA region is important for the activityof the peptide. FIG. 5 shows a graphical representation of the mostefficient peptides of the series. All peptides were tested atconcentration 10⁻⁶M.

Ex Vivo Characterization

Vasomotricity studies were also performed on API-101 alanine scanpeptides (see example 1 for experimental protocol). FIG. 6 shows thatAPI-101 (SEQ ID NO: 1), −101.1, −101.3 and −101.6 (SEQ ID NOs: 2, 4 and7, respectively) all reversed the vasodilation induced by IL-1β (75ng/ml) and that API-101.1 (SEQ ID NO: 2) showed a slightly increasedinhibitory activity over API-101, and abolished 70% of the vasodilation.

Overall, the mutations or substitutions did not significantly increasethe activities of the peptide derivatives over that of API-101 (SEQ IDNO: 1), but information about an important region for the activity ofthe peptide was obtained.

EXAMPLE 3 Effect of Further Optimization of API-101 on the Improvementof its Activity

To further improve the activity and to validate the alanine scanconclusions obtained on the region in API-101 important for itsactivity, the amino acids from the N-terminal end of the peptide weregradually truncated. FIG. 7 shows the sequence of the new peptides aswell as the general pattern of optimization employed for API-101.

In Vitro Characterization:

IL-1β induces proliferation of human fibroblasts cells. Truncatedpeptides were assayed for IL-1β induced WI-38 (human lung fibroblasts)proliferation with the tritiated thymidine uptake protocol (see protocolexample 1).

Relative to API-101 (SEQ ID NO: 1) which abolished 65% of IL-1R inducedproliferation; API-101.10 (SEQ ID NO: 10) and API-101.11 (SEQ ID NO: 11)abolished 100% of IL-1B-induced proliferation (FIG. 8).

Determination of IL-1-induced PGE₂ synthesis was also performed onAPI-101 truncated derivatives. FIG. 18 shows a summary of the potency aswell as the efficacy of the different peptides. API-101.10 (SEQ ID NO:10) was the most efficient and potent truncated peptide with 0.2 nM and1.2 nM IC₅₀ on WI-38 and endothelial cells compared to API-101 (790 nMand 220 nM). API-101.11 (SEQ ID NO: 11) and 12 (SEQ ID NO: 12) showed adecrease in potency and efficacy, which indicated that the peptidetruncation after the arginine influenced the potency and efficacythereof.

Cytotoxicity

Cytotoxicity of the latest derivatives of API-101 was also determined intwo cell types: WI-38 and brain microvascular endothelial cells. Cellviability was assayed as previously described (Beauchamp et al. 2001;Brault et al. 2003). Endothelial and fibroblast cells were incubatedwith peptides at various concentrations at 37° C. for 24 h. MTT(3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) in PBSwas added to the growth medium at a final concentration of 500 μg/ml.Cells with MTT were incubated for 2 h at 37° C. Growth medium was thenaspirated and 200 μl of a solution of 24:1 isopropanol:HCl 1N was addedin each well to lyse the cells. Viable cells transform the MTT product(via the mitochondria) into a measurable colorimetric (blue) productnamed formazan. Formazan production (and cell viability) was determinedby measuring the optical density of 100 μl of lysate at 600 nm.

Cells did not show any toxicity when exposed to 10⁻⁵ M of peptides for24 hours as shown in FIG. 9.

Ex Vivo Characterization

Vasomotricity experiments were also carried out to evaluate the effectof API-101.10 (SEQ ID NO: 10) (the peptide having shown the bestactivity in vitro) on vasodilation induced by IL-1 (for protocol, seeExample 1). API-101.10 (SEQ ID NO: 10) showed the best IC₅₀ at 10.8 nMand was 100 fold more potent than API-101 (182 nM) (FIG. 18). Thepeptides 101.9 (SEQ ID NO: 9), 101.11 (SEQ ID NO: 11), and 101.12 (SEQID NO: 12) showed better IC₅₀ than API-101 (SEQ ID NO:1) (FIG. 18 andFIG. 10). The concentration range for peptides in FIG. 10 was from 10⁻¹⁰M to 10⁻⁵M. Thus, in the ex vivo experiments, API-101.11 (SEQ ID NO: 11)and API-101.12 (SEQ ID NO: 12) showed significantly improved inhibitoryactivities as compared to the parental peptide.

In Vivo Characterization

Systemic Effects of IL-1R/IL-1RAcP Peptides Antagonists

The API-101 derivatives, API-101.10 (SEQ ID NO: 10) (and others) weretested to assess whether they could reverse the physiological actions ofthe natural ligand in vivo by injecting the derivative through thejugular or directly into the stomach (to verify the stability of thepeptide through the digestive tract). Sprague-Dawley rats (300 g) wereanesthetized with isoflurane (2.54%). The natural ligand (IL-1) orvehicle (saline) was injected through the jugular vein (5 μg/kg). Bloodwas taken from the carotid artery for subsequent PGE₂ measurementsbefore and 10 min after each injection. Peptides were administered(dosage based on IC₅₀ values and a volume of distribution equivalent tothe extracellular space) either in the jugular vein or directly in thestomach (5 times dose used intravenously, (iv)). Arterial blood pressureand heart rate were continuously monitored (Gould) while temperature andblood gases (Radiometer) were measured for routine analysis aspreviously described (Li et al. 1997; Hardy et al. 1999; Najarian et al.2000). Experiments were repeated 3 times.

Severe hypotension induced by IL-1β was observed when administered tothe rats by either ways mentioned above. The following peptidesconstitute example antagonists that were able to prevent hypotension invivo:

-   1. API-101.10 (SEQ ID NO: 10): When administrated by jugular    injection after IL-1β injection (5 μg/kg) prevented hypotension by    95% at a concentration of 10⁻⁸M (i.e. it relieved the IL-1    induced-hypotension). Other derivatives like API-101.9 (SEQ ID    NO: 9) were also able to prevent this biological effect of IL-1β but    were less effective than peptides API-101.10 (SEQ ID NO: 10),    101.101 (SEQ ID NO:13) or peptidomimetics, 101.109, 101.111 and    11.112 (FIG. 20) but significantly better than the saline control    (FIG. 11A). This clearly demonstrates that the peptides have an    hypertensive effect in vivo in animals, by reversing the effect of    IL-1β.-   2. When administrated directly into the stomach, at a concentration    of 10⁻⁵M, the peptide reduced IL-1β hypotension by 60%. This result    demonstrated that enteral administration of the 101.10 (SEQ ID    NO: 10) peptide still maintained a major effect on IL-1β induced    hypotension (FIG. 12A) and thus can maintain efficacy and stability    along the digestive tract.

Another way of assessing the effect of IL-1 receptor antagonists of thepresent invention on IL-1R activity in vivo is by measuring PGE₂ levelsin rat serum.

If the IL-1R receptor antagonist of the present invention can preventhypotension in vivo they should be able to prevent also the synthesis ofPGE₂. PGE₂ was therefore measured in serum of rats used for theexperiments mentioned above (e.g. Arterial Blood Pressure variationmeasurement). Examples of results obtained with truncated API-101derivatives peptides is described below.

Once again, API-101.10 (SEQ ID NO: 10) was shown to be the mosteffective of the API-101 derivatives tested in preventing PGE₂ synthesis(60%) when the peptide was injected in the jugular. Higher inhibitionwas obtained when the peptide was injected directly in the stomach(FIGS. 11B and 12B).

Further Optimization of API-101.10

API-101.10 (SEQ ID NO: 10) was identified as the best peptide derivativefrom the last round of optimization. Thus, truncation of API-101 from 9to 7 amino acids from the N-terminal could improve the potency withoutcompromising the efficacy in vitro, ex vivo and in vivo.

FIG. 15 shows the next mutations that were performed on API-101.10 (SEQID NO: 10). The arginine of API-101.10 (SEQ ID NO: 10) was replaced bycitrulline—to change from a guanidine to a urea group near theN-terminal. Other mutations (e.g. E to Q in API-101.102, SEQ ID NO: 14,and 101.103, SEQ ID NO: 15) and a truncated peptide at the C-terminal(API-101.108, SEQ ID NO: 20) were also performed to improve the potencyand the efficacy of the peptides.

FIG. 15 shows an in vitro characterization of these peptides.Measurement of PGE₂ was performed with piglet brain microvesselendothelial cells and WI-38 human fibroblasts. Some of the mutationswere advantageous and gave major increases in potency. For example:API-101.103 (SEQ ID NO: 15), and 101.107 (SEQ ID NO: 19) showed morethan 1000 fold better potency with IC₅₀ of 0.05 μM and 0.1 μM in humanWI-38 cells.

For these newly derived peptides, ex vivo experiments were thenconducted. Brain tissues were incubated with the peptides and IL-1β andcGMP was measured with a commercial kit (Amersham bioscience, cGMP assayBiotrack™ system). API-101.10 (SEQ ID NO: 10) already inhibited 85% ofIL-1β-induced cGMP production (10⁻⁶M) and API-101.103 (SEQ ID NO: 15)and 101.106 (SEQ ID NO: 18) inhibited more than 90% of cGMP production(results not shown). It thus seems, that removal of the negative chargeof the glutamate and removal of the threonine can improve the potency ofthe antagonist. Of note, the activity of API-101.10 (SEQ ID NO: 10) wasshown to be superior to that of the Amgen drug Kineret™ (data notshown).

Taken together, the present invention clearly demonstrates that API-101is a potent and efficacious IL-1 receptor antagonist. Furthermore, itclearly demonstrates that starting from API-101 (SEQ ID NO: 1), theinventors could derive, in a systematical fashion, even more potent andefficacious antagonists (as shown by a comparison of the IC₅₀ of API-101and that of derivatives of the 101.100 series). The present inventiontherefore provides the means to identify new IL-1R/IL-RacP receptorantagonists and methods of treating or preventing diseases or disordersassociated with a defect in the pathway involving IL-1R/IL-RacP. Theperson of ordinary skill in the art can also derive peptidomimetics andother derivatives based on the teaching of the present invention and thestate of general knowledge in the art, and as described below.

EXAMPLE 4 Efficacy of ApI-101 in a Rat Model of Inflammatory BowelDisease (IBD)

IBD is a chronic inflammation of the gastrointestinal tract with highincidence among the human population. The present experiment was done inorder to verify if the peptide API-101.10 (SEQ ID NO: 10) could preventyet another inflammatory process in an IBD animal model induced with thetrinitrobenzene sulphonic acid (TNBS). TNBS causes an IL-12 mediatedTH-1 response characterized by transmural infiltration of neutrophilsand macrophage, fissuring ulcerations and submucosal fibrosischaracteristic of acute intestinal inflammation and Crohn's disease(Bouma and Strober 2003).

Inflammatory Bowel Disease Model:

Colon inflammation was induced by intra-rectal/colon administration ofthe hapten trinitrobenzene sulphonic acid (TNBS) on male Sprague-Dawleyrats (175-200 g) (Bouma, Nature Rev, 2003; Morris, Gastroenterology,1989). Animals were anesthetized with isoflurane and TNBS dissolved in50% ethanol (vol/vol). 120 mg/ml (TNBS) was administered into the colon(total volume of 0.25 ml per rat) using a polyethylene tube (PE50). Thecannula was inserted at 8 cm from the anus and kept in place for atleast 15 min after TNBS administration in order to prevent expulsion ofthe solution. Two hours prior to TNBS administration, The API-101derivative API-101.10 (SEQ ID NO: 10)1.1 mg/kg) or 0.9% saline wasadministered intravenously via the caudal vein (total volume of 0.3 ml).API-101.10 (2.2 mg/kg, 6 times dose used for blood pressure experimentsbased on t½=2-3 h for various peptides) or 0.9% saline were thencontinuously infused using primed intraperitoneal alzet pumps. A thirdgroup (control) was not injected with TNBS. Six days afteradministration of TNBS, rats were killed by CO₂ inhalation. Day 6 waschosen as an endpoint because by day 7 spontaneous tissue regenerationbegins and this can mask the therapeutic effect of the tested peptide orpeptide derivative. Colon was removed and examined macroscopically(adhesions, ulcerations, discoloration and bleeding) and histologically(neutrophil infiltration, epithelial injury, crypt distortion andulcerations) (Anthony et al. 1995; Padol et al. 2000; Dieleman, L A, etal. 1997; Torres M I et al. 1999). Two animals per group were studied.

Histological transversal sections were cut at 4-6 cm from the proximalanal region and colored with the hematoxylin/eosin method.

The TNBS model of inflammatory bowel disease reproduces the inflammatorycharacteristics and tissue injuries of Crohn's disease (e.g. in humans).As showed in FIGS. 14 A and B, morphologically, the colon of the animalsinjected with TNBS presented thickening, edema and discoloration of theintestinal wall indicating a significant inflammation. Macroscopiccharacteristics of colons from animals pre-treated with API-101.10 (SEQID NO: 10) resemble those of the control animals (FIG. 14 C).Histological features consisted of neutrophil infiltration into theepithelial layer and crypts (see FIG. 16 B), epithelial lining injury aswell as the loss of crypts (FIG. 16 B). Pre-treatment of the animalswith API-101.10 (SEQ ID NO: 10) prevented TNBS-induced colon damage. InFIG. 16, one can appreciate that the organization and integrity of thecrypts in the API-101.10 (SEQ ID NO: 10)-treated colon is conserved evenif there is still some inflammation (half the dose of API-101.10 wasused as compared to the macroscopic analysis experiment). The injurieson the epithelium lining shown in FIG. 16 B are completely prevented inthe API-101.10 (SEQ ID NO: 10) treated animals (FIG. 16 C). Hence, theIL-1R antagonists of the present invention are also extremely efficientin an animal model of inflammatory bowel disease.

EXAMPLE 5 Peptidomimetics API-101.109. API-101.110

To further improve the efficacy and the potency of the antagonists ofthe present invention, peptidomimetics were synthesized and screened invitro. In one embodiment, the peptidomimetics are derived fromAPI-101.10 (SEQ ID NO: 10) or API-101.107 (SEQ ID NO: 19) and theprimary structures are: for API-101.109 RY(HyVal)PELA (FIG. 20) and forAPI-101.110 RY(I²aa)ELA (FIG. 21) where HyVal is beta-Hydroxyvaline andI²aa is indolizidin-2-one amino acid(2-oxo-3-amino-azabicyclo[4.3.0]nonane-9-carboxylic acid. Thesepeptidomimetics are also D-peptides.

Methodology: Preparation of Solid Support

Benzhydrylamine resin hydrochloride (2 g, Advanced Chemtech, Lot #11988, 100-200 mesh, loading 1.2 mmol/g) was washed for one min threetimes with 10 ml/g of each of the following reagents: 5% DIEA/CH₂Cl₂;CH₂Cl₂; DMF. The resin was treated with a solution ofN-(Fmoc)aminocaproic acid (1.27 g, 3.6 mmol, 150 mol %), TBTU (1.27 g,3.96 mmol, 165 mol %), DIEA (690 μL, 3.96 mmol, 165 mol %), and HOBt(535 mg, 3.96 mmol, 165 mol %) in DMF (20 ml, 10 ml/g of resin), andagitated for 1 h when a negative Kaiser test was observed. The resin waswashed with 10 ml/g of the following solutions in an alternatingsequence: DMF (3×1 min) and isopropyl alcohol (3×1 min). The resin wasthen treated with piperidine in DMF (20% v/v, 20 ml, 1×2 min, 1×3 min,1×10 min), followed by an alternating sequence of 10 ml/g of DMF (3×1min) and isopropyl alcohol (3×1 min). The resin was agitated with asolution of 4-[(R,S)-α-1(9H-fluoren-9-yl)-methoxy-formamido]-2,4-dimethoxybenzyl]-phenoxyaceticacid (Knorr linker, 1.94 g, 3.6 mmol, 150 mol %), TBTU (1.27 g, 3.96mmol, 165 mol %), and DIEA (690 μL, 3.96 mmol, 165 mol %) in DMF (20 ml)for 1 h. The resin was sequentially washed with 10 ml/g of the followingsolutions: DMF (3×2 min), isopropyl alcohol (3×2 min), and CH₂Cl₂ (3×2min). Drying of the resin under high vacuum overnight yielded 3.66 gresin.

Determination of Loading

Piperidine (20 g) and DMF (20 g) were mixed. To a quantity of thissolution (20 ml, 18.08 g) in a sample vial was added dry resin (20 mg),and the suspension gently agitated by passage of a stream of argon.After 50 min, the resin was allowed to settle. An aliquot of solution (1ml) was diluted 50-fold with ethanol, and the absorbance measured at 301nM [(N-(9-fluorenyl-methyl)piperidine UV λ_(max)-267 nM (ε 17500), 290(5800) and 301 (7800)]. Two separate determinations (averaged) gaveA₃₀₁=0.0785. The following equation: [c (mmol/g)=(OD×50×10²)/7800] gavec=0.50 mmol/g (Meienhofer et al. 1979).

Peptide Synthesis

Amino acids were purchased from Advanced Chemtech (Louisville, Ky.), andused as the following derivatives: N-Fmoc-D-Ala-OH.H₂O, N-Fmoc-D-Leu-OH,N-Fmoc-D-Glu(O-t-Bu)-OH, N-Fmoc-D-Pro-OH, N-Fmoc-D-Tyr(O-t-Bu),N-Fmoc-D-Arg(Pmc)-OH. (R)-β-hydroxy-N-(Fmoc)valine was prepared from(R)-β-hydroxy-N-(Boc)valine (Dettwiler et al. 2003) by removal of theBoc:group (1:1 TFA(trifluoroacetic acid)/CH₂Cl₂), protection withFmoc-OSu and NaHCO₃ in aqueous acetone (Capatsanis et al. 1983),followed by purification by chromatography over silica gel (1:1:98MeOH/HOAc/CHCl₃) and lyophilization from aqueous acetonitrile (78%yield).(3R,6R,9R)-2-Oxo-3-[N-(Fmoc)amino]-1-azabicyclo[4.3.0]-nonane-9-carboxylicacid was prepared from (3R,6R,9R)-methyl2-oxo-3-amino-1-azabicyclo[4.3.0]-nonane-9-carboxylate (in turn prepared(Lombart et al. 1996) from D-glutamic acid) by Fmoc-protection withFmoc-OSu and NaHCO₃ in aqueous acetone (Capatsanis et al. 1983),followed by selective hydrolysis of the methyl ester (Pascal et al.1998). Peptide synthesis was performed on a 0.1 mmol scale (200 mgresin), and conducted by deprotection with piperidine in DMF (10 ml/gresin, 20% v/v, 1×2 min, 1×3 min, 1×10 min) followed by washing with DMF(10 ml/g resin, 5×1 min). Fmoc protected amino acid (0.5 mmol, 500 mol%) dissolved in a solution of TBTU in DMF (0.25 M, 2 ml) was added tothe resin. After agitation of the resin (5 min), DIEA (0.6 mmol, 600 mol%) was added, and agitation continued for 1 h. The resin was washed withDMF (10 ml/g resin, 5×1 min), and coupling efficiency determined usingthe Kaiser test. The resin was agitated using a mechanical vortexapparatus during coupling, rinsing and deprotection sequences. Rp-HPLCanalysis was performed on an Alltech C18 column (dimensions 250 mm×4.6mm) using acetonitrile/water/TFA mixtures, where solvent A=water/0.1%TFA and solvent B=MeCN/0.1% TFA (see below). The flow rate was 0.5ml/min, and detection was performed at 214 nM.

Peptidomimetic API-101.109 (KH-C29099)

Cleavage from the resin (180 mg) with simultaneous side chaindeprotection was conducted by treating the resin with 20 ml/g of acocktail containing TFA (82.5%), thioanisole (5%), water (5%), phenol(5%) and triethyl silane (2.5%) and agitating with a mechanical vortexapparatus for 1 h at room temperature. Subsequent filtration, rinsingwith TFA (2×1 ml) and precipitation in Et₂O at 0° C. gave the peptide.Isolation of the crude peptide as the dihydrochloride salt bylyophilization from HCl solution (1 M) gave a white powder (18 mg) thatwas shown to be >90% pure by rp-HPLC (RT=14.6 min) using an eluant of540% B in A over 20 min. LRMS calcd for C₃₉H₆₄N₁₁O₁₁ (MH⁺) 862, found862.

Peptidomimetic API-101.110 (KH-C50110)

Cleavage from the resin (22 mg) with simultaneous side chaindeprotection was conducted by treating the resin with 20 ml/g of acocktail containing TFA (82.5%), thioanisole (5%), water (5%), phenol(5%) and triethyl silane (2.5%) and agitating with a mechanical vortexapparatus for 1 h at room temperature. Subsequent filtration, rinsingwith TFA (2×1 ml) and precipitation in Et₂O at 0° C. gave the peptide.Isolation of the crude peptide as the dihydrochloride salt bylyophilization from HCl solution (1 M) gave a white powder (5.7 mg) thatwas shown to be >85% pure by rp-HPLC (RT=19.8 min) using an eluant of540% B in A over 20 min. LRMS calculated for C₃₈H₆₀N₁₁O₁₀ (MH⁺) 830,found 830.

Results:

-   IL-1-induced PGE₂ synthesis assay on endothelial cells was used as a    screening assay for the peptidomimetics. The peptidomimetic compound    API-101.110 (FIG. 21) had a potency of 0.2 pM of IC₅₀, which is 10    fold higher than API-101.107 (SEQ ID NO: 19) with twice the efficacy    of the later. The compound API-101.109 (FIG. 20) also showed an    improved potency in inhibiting PGE₂ (IC₅₀) (FIG. 19) but its K_(D)    is to high to be an efficient drug.

EXAMPLE 6 Efficacy of API-101.10, API-101.107 and API-101.113 in a RatModel of Inflammatory Bowel Disease (IBD)

Further experiments were carried-out in order to verify if lead peptidesTTI-101.10, previously termed API-101.10 (SEQ ID NO: 10), TTI-101.107and TTI-101.113 (also termed 101.107 and 101.113, respectively; orAPI-101.107 and API-101.113, respectively) could prevent theinflammatory features on the animal IBD model described in Example 4.Colon inflammation was induced by intra-rectal/colon administration ofthe hapten trinitrobenzene sulphonic acid (TNBS) as described in Example4. Two hours prior to TNBS administration, peptides, peptidomimetics or0.9% saline were administered intravenously (iv) via the caudal vein(various concentrations of mg/kg/d) (total volume of 0.3 ml). Forcontinuous infusion, API-101.10 (or other peptides orpeptidomimetics)(2.2 mg/kg, 4 times dose used for blood pressureexperiments based on t½=2-3 h of various peptides) or 0.9% saline werethen continuously infused using primed intraperitoneal alzet pumps. Athird group (control) was not injected with TNBS. For intermittentadministration, fifteen minutes after TNBS administration, 101.10(0.25-1 mg/kg), 101.107 (0.2 mg/kg), 101.113 (0.05-1 mg/kg) wereadministered by intermittent intraperitoneal injection (ip). Also, theseIL-1R antagonists were given twice a day (BID); Remicade® (anti-TNFα)(10 mg/kg) and dexamethasone (0.75 mg/kg) were administeredintraperitoneally but only once a day (qd). The intrarectaladministration (ir) of 101.10, 101.113 (1 and 2.5 mg/kg), and 5-ASA (50mg/kg) was done one hour after TNBS administration, and twice a day,except for 5-ASA which is once a day. Finally, 101.10 (1-5 mg/kg) wasalso administered orally by gavage (po), twice a day. Forty-eight hoursafter administration of TNBS, rats were killed by CO₂ inhalation. Colonwas removed and assessed macroscopically (adhesions, ulcerations,discoloration and bleeding) and histologically (neutrophil infiltration,epithelial injury, crypt distortion and ulcerations). One to sevenanimals per group were studied, according to treatments. Myeloperoxidase(MPO) activity was measured on tissue lysates.

Results:

As mentioned previously, The TNBS animal model is a valid in vivo modelfor inflammatory disease in humans, and more particularly ofinflammatory bowel disease (IBD) since it reproduces the inflammatorycharacteristics and tissue injuries of Crohn's disease. As shown inTable 2, intraperitoneal continuous and intermittent injections ofantagonists of the present invention (e.g. peptides, peptide derivativesand peptidomimetics) at different dosage prevented tissue damages due toinflammation such as formation of ulcers, loss of crypts and epitheliumlining injury.

Animals that received intraperitoneal osmotic pumps (continuousinfusion) containing 101.10 and 101.107 demonstrated marked reductionsin MPO activity, macroscopic and histologic score, superior orequivalent in efficacy to Kineret (Table 2). Intermittent administrationof 101.10, 101.107 (one concentration only) and 101.113 revealed adose-dependent efficacy of twice a day administration which surpassesthat observed with currently utilized agents for IBD, namelydexamethasone, Remicade® and 5-ASA. Macroscopic observation of colonicinjuries were scored (4 blinded observers) and animals treated withpeptides (BID) presented less adhesions and ulcerations (less than 50%compare to TNBS-treated animals). Animals also looked considerably morevigorous.

TABLE 2 Summary of in vivo results macroscopic histologic evaluationevaluation dose MPO (% of (median (median Treatment (mg/kg/d) n = TNBS +Saline) score) score) TNBS + Saline 120 mg/ml 6 100 2/2 5/5 Continuousinfusion (ip) TNBS + 101.10 0.25 2 34 nd 2 TNBS + 101.10 0.75 2 54 nd4.4 (n = 1) TNBS + 101.10 2.2 3 46 ± 22 nd 2 (2) TNBS + 101.10 4.0 2 82nd 2 TNBS + 101.107 0.5 2 37 nd 1.5 TNBS + Kineret 8.0 2 63 nd 2Intermittent injection ip) TNBS + 101.10 0.25 - BID 2 85 0.87 2.25TNBS + 101.10 0.50 - BID 2 126 0.87 3 TNBS + 101.10 1.0 - BID 6 47 ± 9 0.8 ± 0.1 1.25 (1-2.8)** TNBS + 101.10 1.0 - qd 2 67 1.63 5 TNBS +101.10 1.0 - BID 7 123 ± 18* 1.1 ± 0.1 2.6 (12 h after TNBS) TNBS +101.107 0.2 - BID 2 112 1.37^(†) 2.5 TNBS + 101.113 0.05 - BID 1 571.25^(†) nd TNBS + 101.113 0.2 - BID 2 112 1.13 2.6 TNBS + 101.113 0.5 -BID 1 45 0.75^(†) nd TNBS + 101.113 1.0 - BID 1 67 1.75 nd TNBS + Saline120 mg/ml 6 100 2/2 5/5 Intermittent injection (ip) TNBS + Remicade10.0 - qd 3 88 ± 41 0.5 2.5 (1-3.75)** TNBS + Dexamethasone 0.75 - qd 260 0.87 3 Intrarectal administration TNBS + 101.10 + PEG-400 1.0 - BID 2110 1.25t 3.2 TNBS + 101.10 2.5 - BID 6 111 ± 25  1.4 ± 0.1 nd TNBS +101.10 + PEG-400 2.5 - BID 2 59 1.17^(†) 3.9 TNBS + 101.113 1.0 - BID 131 1.5 nd TNBS + 101.113 2.5 - BID 1 20 1.75 nd TNBS + 5-ASA 50.0 - qd 249 1.5 3.3 Oral administration TNBS + 101.10 5.0 - BID 1 65 0.75 2.25TNBS + 101.10 + PEG-400 1.0 - BID 2 167 1.0 3.5 TNBS + 101.10 + PEG-4002.5 - BID 2 176 1.25 3.5 TNBS + 101.10 + PEG-400 5.0 - BID 2 57 1.5 3.6nd: not determined *Note: leucocyte infiltration has already occurred**Range ^(†)Animals were considerably more vigorous

TABLE 3 Histological Injury Scoring System Score No injury 1 Small ulcer(<5 crypts) 2 Medium ulcer (5-10 crypts) 3 Large ulcer (10-20 crypts) 4Marked denudation (>20 crypts) 5 (adapted from Peterson et al., Dig DisSci, 2000)

FIG. 22 shows a graphical representation of the macroscopic scoring ofevery peptide for one particular concentration and the list of theevaluated features. Also, as may be seen in FIG. 23, animals treatedwith intermittent injections of peptides presented 20 to 50% lessneutrophil infiltrations (myeloperoxidase assay) as compare to the TNBScontrol. Examination of histologic sections revealed thatpeptide-treated animals presented less characteristics of inflammationinduced-colonic injury. The histological injury scoring system used isshown in Table 3, and the results are schematized in FIG. 24. Of course,other scoring systems could be used and adapted by the skilled artisanto which the present invention pertains. Hence, one may appreciate thereduction in the amount of ulcers and epithelial lining injury in PanelC and D of FIG. 25, as compared to Panel B representing the inflamedtissue. Furthermore, administration of the agents of the presentinventions (e.g. peptide) 12 hours after the TNBS induction resulted inreduction of colonic inflammation also. The high myeloperoxidaseactivity remaining is due to the fact that neutrophil infiltration hadalready occurred before treatment.

In order to demonstrate that the peptide, and peptidomimetics of thepresent invention, can be administered by other means and reduce theinflammation generated with the TNBS, API-101.10 and API-101.113 wereinjected intrarectally. The inflammation level was assayedmacroscopically and histologically as above. As may be seen in Table 2API-101.10 at 2.5 mg/kg/d reduced substantially (50%) the MPO activityand partially prevented colonic tissues damages.

API-101.10 was also administrated by another means: gavage (twice a day)to demonstrate the stability of the peptide through the digestive tract.At the concentration of 5.0 mg/kg/d API-101.10 substantially reduces theinflammatory features as well as the MPO activity, thereby validatingthe stability of the compounds of the present invention.

EXAMPLE 7 TTI-101.107 Peptide Derivatives and Mimics

Using TTI-101.107 (SEQ ID NO: 19, and FIG. 15; IC₅₀ of 1.2 pM) as a leadpeptide, several series of analogs were designed, synthesized and testedto establish the importance of each residue.

Structure vs Activity:

As may be seen in Table 4, when the terminal D-arginine was acetylatedto give compound TTI-101.121 (SEQ ID NO: 29), the activity of thepeptide was completely lost. On the other hand, the arginine residue maybe replaced with ornithine or lysine and the resulting peptide maintainsits activity (TTI-101.114, SEQ ID NO: 22; and TTI-101.115, SEQ ID NO:23). It thus seems that the guanidine group of arginine (as withornithine) may be important for peptide activity.

From data obtained by the replacements at the D-Threonine and D-valineresidues previously shown above (see FIGS. 15 and 19) using peptidesTTI-101.105 (SEQ ID NO: 17), and 101.106 (SEQ ID NO:18), andpeptidomimetic TTI-101.109, a potential for a turn region about theseresidues was hypothesized and two peptides mimics were generated byintroducing both (3R,6R,9R; TTI-101.110) and (3S,6S,9S;TTI-101.112)-indolizadin-2-one amino acids (R- and S-I2aa). Thesepeptidomimetics shown in FIG. 26, mimic type II and type II′ beta-turns,respectively. Peptidomimetic TTI-101.110 exhibited an activity of 10 pM(FIG. 26), comparable to that of peptide 101.107 from which it isderived.

The importance of the glutamate position was addressed using TTI-101.117(SEQ ID NO: 25), TTI-101.118 (SEQ ID NO: 26) and TTI-101.123 (SEQ IDNO:31), in which glutamate is replaced with aspartate, asparagine andalanine, respectively. The results show (Table 4) that removing thecarboxylate or carboxamide is deleterious for peptide function.

Examining the C-terminal D-leucinyl-D-alanine residues, a series ofderivatives with deletions and substitutions were generated: TTI-101.113(SEQ ID NO: 21); TTI-101.119 (SEQ ID NO: 27) and TTI-101.120 (SEQ IDNO:28). Deletion of the D-alanine residue gives rise to hexapeptideTTI-101.113 ((SEQ ID NO:21) Table 4) having a 7-30 μM activity.Modification of the leucine residue resulted in loss of activity range.

Based on the data shown above, two other mimetic compounds weresynthesized (see FIG. 26): TTI-101.124 (ry[R-I2aa]el which showed anIC₅₀ of 2.4 μM and an efficacy of 100%; FIG. 26) and TTI-101.125((D-orn)y[R-I2aa]ela which showed an IC₅₀ of 90 μM and 100% efficacy;FIG. 26).

Derivatives of 101.113 Peptide

Based on lead peptides (101.107 rytpela (SEQ ID NO: 19), 101.10 rytvela(SEQ ID NO: 10) and 101.113 rytpel (SEQ ID NO: 21), another series ofanalogs was made to examine further the structure-activity(structure-function relationship) relationship of the peptides andderivatives.

Exploring the importance of the basic amino acid terminal arginine, aseries of analogs have shown that the activity was relatively diminishedwhen the guanidine portion was replaced by a basic amine. Indeed,compounds TTI-101.126 (SEQ ID NO: 32), TTI-101.133 (SEQ ID NO: 37) andTTI-101.134 (SEQ ID NO: 38) exhibited little or no activity (Table 5).When the stereochemistry was inverted as in TTI-101.135 (SEQ ID NO: 39),in which the arginine “R” is an L-amino acid, as opposed to a D-aminoacid in SEQ ID NO:21, the activity was lowered but not lost completely(Table 5).

As shown further in Table 5, the activity of the peptide 101.113 wasalso relatively decreased when the aromatic residue tyrosine, with itsphenolic group, was replaced with aromatic residue phenylalanine(101.132; SEQ ID NO:36) or tryptophan (101.128; SEQ ID NO: 34). Theremoval of the hydroxyl group in TTI-101.127 (SEQ ID NO: 33) completelyabolished the activity of the peptide, but the replacement of tyrosinewith tryptophan lowered, but yet maintained the activity.

Replacing the C-terminal leucine by valine also caused a decrease inactivity, demonstrating an importance of the length of the hydrophobicresidue, as may be observed in Table 5 with TTI-101.129 (SEQ ID NO:35)(rytpev 400 nM; 50%).

Based on the lead peptidomimetic (TTI-101.125; FIG. 26) another seriesof mimetics was prepared to explore yet further the structure-activityof the compounds of the present invention.

Using aza-amino acid residues to respectively replace the tyrosine, theleucine and alanine residues, the series 101.136 to 101.140 and101.141-101.144 were prepared (FIGS. 27 and 28). Because aza-amino acidscan improve the resistance of peptides to enzymatic degradation, themaintenance of the activity in certain analogs exemplifies one means forincreasing the duration of their in vivo action. These lastmodifications led to the development of compound TTI-101.140. FIGS. 29and 30 show the structure and activity of the peptidomimetics 101.125,101.136-101.144 and in particular the potency and efficiency of mimetic101-140 which showed an increased activity.

TABLE 4 IL-1 β-induced proliferation and PGE₂ synthesis in presence ofpeptidomimetics Proliferation in PGE2 synthesis in TF-1 cellsendothelial cells (human) (porcine) Peptides Sequence IC₅₀ Emax (%) IC₅₀Emax (%) 101.113 rytpel 30 pM 100 7.4 pM  80 101.114 kytpela nd nd  2 pM50 101.115 [orn]ytpela ~1 pM 100 nd nd 101.116 rwtpela 0.5 nM   75 13 pM45 101.117 rytpdla nd nd 10 pM 100  101.118 rytpqla nd nd nd nd 101.119rytpefa nd nd nd nd 101.120 rytpema nd nd nd nd 101.121 [Ac]rytpela ndnd nd nd 101.122 rytpepa nd nd nd nd 101.123 rytpala nd nd nd nd

TABLE 5 Characterization of 101.113 peptide derivatives IL-Iβ-inducedTF-1 proliferation Peptide Sequence IC50 Emax (%) 101.113 rytpel 7 pM 70101.126 [Orn]ytpel * 0 101.127 rfvpela nd <30 101.128 rwtpel 3 nM 100101.129 rytpev 400 nM 50 101.132 rftpel 4 nM 35 101.133 kytpel nd <10101.134 [Cit]ytpel 10 μM 10 101.135 Rytpel 2 nM 63 * Could not bedetermined The “R” denotes an L-aa

CONCLUSIONS

In summary, the present invention describes efficient and potentantagonists of IL-1R/IL-1RacP receptor that can abrogate the biologicaleffects of the interleukin-1 both in vitro, ex vivo and in vivo. Thesepeptides were effective in vitro on different cell types and againstdifferent biological effects (proliferation and PGE₂ synthesis), ex vivoby abolishing the vasomotor effect of IL-1 on pial vessels and PGE₂synthesis on fresh sample tissues. Moreover these API-101 derivativeswere also very effective in vivo when administrated systemically anddirectly into the stomach. The last result obtained with the stomachdelivery show that the peptides of the present invention have thepotential of being active when administered orally. This was indeeddemonstrated when gavage was used. More importantly, in an establishedrat model of Inflammatory Bowel Disease (IBD), API-101.10 could preventcolon damages induced by injection of TNBS in rat. Based on the resultswith other derivatives (in the 101.100 series), it has been demonstratedthat these peptide derivatives and peptidomimetics thereof are potentanti-inflammatory agents, as demonstrated by their role in preventingcolon damage induced by injection of TNBS in rat.

The present invention shows that the peptides and peptidomimetics of thepresent invention have clear effects on a number of pathways involvingIL-1R/IL-1RacP. The therapeutic and prophylactic potential of thepresent invention has therefore broad impacts for animals in general andmore particularly for mammals and especially for humans.

Based on the disclosure herein, those skilled in the art can developpeptides, peptide derivatives and peptidomimetics screening assays whichare useful for identifying further IL-1R/IL-1RacP receptor inhibitingcompounds or improve those exemplified herein. The assays of the presentinvention may be developed for low-throughput, high-throughput, orultra-high throughput screening formats. Of course, assays of thepresent invention include assays that are amenable to automation.

Thus, working with peptides possessing natural amino acids, the presentinvention demonstrate that they are potent and efficacious compounds invitro and in vivo studies (e.g. TTI-101.107 (SEQ ID NO: 19 and 113 [SEQID NO:21]). Furthermore, the present invention provides an initialdescription of the pharmacophore and conformation necessary for activityand the peptides and mimetics derived therefrom.

Of note, the invention provides a hexapeptide lead: TTI-101.113 (rytpel([SEQ ID NO:21] having a very significant activity (7.4 μM; 80%efficacy). Further mimics or mimetics were generated with indolizadinoneamino acid to replace the central D-threonine-d-valine(D-Pro) regionthereby enabling the identification of lead mimics TTI-101.125 andTTI-101.140.

Of course the combination of antagonists of the present invention or acombination thereof with known drugs could further increase the medical,clinical and drug development potential of the present invention.

Although the present invention has been described hereinabove by way ofillustrative embodiments thereof, it will be appreciated by one skilledin the art from reading of this disclosure that various changes in formand detail can be made without departing from the spirit and scope ofthe invention as defined in the appended claims.

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1-38. (canceled)
 39. A method of treating a disease, disorder orcondition associated with IL-1 or IL-1R function in a subject, themethod comprising administering a peptide into the subject in need oftreatment, wherein the peptide is 5 to 25 amino acids long and containsan amino acid sequence that includes at least four amino acids fromRYTVELA (SEQ ID NO: 10) and wherein the at least four amino acidsmaintain their relative positions as they appear in RYTVELA (SEQ ID NO:10).
 40. The method of claim 39, wherein the peptide is 5 to 16 aminoacids long.
 41. The method of claim 39, wherein the peptide is 5 to 9amino acids long.
 42. The method of claim 39, wherein the amino acidsequence includes at least five amino acids from RYTVELA (SEQ ID NO: 10)and wherein the at least five amino acids maintain their relativepositions as they appear in RYTVELA (SEQ ID NO: 10).
 43. The method ofclaim 39, wherein the peptide is 6 to 25 amino acids long and the aminoacid sequence includes at least six amino acids from RYTVELA (SEQ ID NO:10) and wherein the at least six amino acids maintain their relativepositions as they appear in RYTVELA (SEQ ID NO: 10).
 44. The method ofclaim 39, wherein the amino acid sequence is selected from the groupconsisting of: RYTPEL (SEQ ID NO:21), RYTVQLA (SEQ ID NO:15), KYTPELA(SEQ ID NO:22), RYTPDLA (SEQ ID NO:25), RYTVELA (SEQ ID NO:10), RYVVELA(SEQ ID NO:18), RWTPELA (SEQ ID NO:24), RYTVEL (SEQ ID NO:20), RYTPEL(SEQ ID NO:39), RWTPEL (SEQ ID NO:34), PRYTVELA (SEQ ID NO:9), APRYTVALA(SEQ ID NO:7), RYTPEV (SEQ ID NO:35), YTVELA (SEQ ID NO: 11), TVELA (SEQID NO: 12), and RFTPEL (SEQ ID NO:36).
 45. The method of claim 39,wherein the peptide comprises one or more modifications to increaseprotease resistance, serum stability and/or bioavailability.
 46. Themethod of claim 45, wherein the one or more modifications are selectedfrom N- and/or C-terminal acetylation, glycosylation, biotinylation,substitution with D-amino acid, or un-natural amino acid, and/orcyclization of the peptide.
 47. The method of claim 46, wherein thepeptide contains at least one D-amino acid.
 48. The method of claim 39,wherein the disease, disorder or condition is an acute or chronicinflammatory disease, disorder or condition.
 49. The method of claim 39,wherein the disease, disorder or condition is selected from rheumatoidarthritis, Crohn's disease, ulcerative colitis, osteoarthritis,psoriasis, inflammatory bowel disease, septic shock, stroke,encephalitis, respiratory distress syndrome, and/or meningitis.
 50. Themethod of claim 39, wherein the peptide is administered orally.
 51. Amethod of treating a disease, disorder or condition associated with IL-1or IL-1R function in a subject, the method comprising administering apeptide into the subject in need of treatment, wherein the peptide is 5to 25 amino acids long and contains an amino acid sequence that is atleast 70% identical to RYTVELA (SEQ ID NO: 10).
 52. The method of claim51, wherein the amino acid sequence is at least 85% identical to RYTVELA(SEQ ID NO:10).
 53. The method of claim 51, wherein the peptide is 5 to16 amino acids long.
 54. The method of claim 51, wherein the peptide is5 to 9 amino acids long.
 55. A method of treating a disease, disorder orcondition associated with IL-1 or IL-1R function in a subject, themethod comprising administering a peptide into the subject in need oftreatment, wherein the peptide is a non-competitive inhibitor of theIL-1 receptor and contains an amino acid sequence that includes at leastfour amino acids from RYTVELA (SEQ ID NO: 10) and wherein the at leastfour amino acids maintain their relative positions as they appear inRYTVELA (SEQ ID NO: 10).
 56. A method comprising administering a peptidedetermined to be a non-competitive inhibitor of the IL-1 receptor into acell system expressing the IL-1 receptor thereby inhibiting, reducing orpreventing an IL-1 associated function in the cell system, wherein thepeptide contains an amino acid sequence that includes at least fouramino acids from RYTVELA (SEQ ID NO:10) and wherein the at least fouramino acids maintain their relative positions as they appear in RYTVELA(SEQ ID NO: 10).
 57. The method of claim 56, wherein the cell system isan in vitro system.
 58. The method of claim 56, wherein the cell systemis an ex vivo system.
 59. The method of claim 56, wherein the cellsystem is an in vivo system.
 60. The method of claim 59, wherein the invivo system is an animal model.
 61. The method of claim 59, wherein thein vivo system is a human subject.
 62. The method of claim 56, whereinthe IL-1 associated function comprises PGE₂ synthesis.
 63. The method ofclaim 56, wherein the IL-1 associated function is involved in aninflammatory reaction.
 64. The method of claim 56, wherein the peptideis 5 to 25 amino acids long.
 65. The method of claim 56, wherein thepeptide is 5 to 16 amino acids long.
 66. The method of claim 56, whereinthe peptide is 5 to 9 amino acids long.
 67. A peptide that is 5 to 25amino acids long and has an amino acid sequence that includes at leastfour amino acids from RYTVELA (SEQ ID NO: 10) and wherein the at leastfour amino acids maintain their relative positions as they appear inRYTVELA (SEQ ID NO: 10), and further wherein the peptide contains one ormore D-amino acids.
 68. The peptide of claim 67, wherein the amino acidsequence includes at least five amino acids from RYTVELA (SEQ ID NO: 10)and wherein the at least five amino acids maintain their relativepositions as they appear in RYTVELA (SEQ ID NO: 10).
 69. The peptide ofclaim 67, wherein the peptide is in natural or in inverse configuration.70. The peptide of claim 67, wherein the peptide contains a mixture ofL-amino acid and D-amino acid.
 71. A peptide that is 5 to 25 amino acidslong and has an amino acid sequence that is at least 70% identical toRYTVELA (SEQ ID NO: 10), and wherein the peptide contains one or moreD-amino acids.